U.S. patent application number 15/376321 was filed with the patent office on 2017-08-31 for methods and compositions for enhanced delivery of compounds.
The applicant listed for this patent is The Salk Institute for Biological Studies, Sanford Burnham Prebys Medical Discovery Institute. Invention is credited to Lilach Agemy, Dinorah Friedmann-Morvinski, Venkata Ramana Kotamraju, Erkki Ruoslahti, Kazuki Sugahara, Inder Verma.
Application Number | 20170246236 15/376321 |
Document ID | / |
Family ID | 44120964 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170246236 |
Kind Code |
A1 |
Agemy; Lilach ; et
al. |
August 31, 2017 |
METHODS AND COMPOSITIONS FOR ENHANCED DELIVERY OF COMPOUNDS
Abstract
Disclosed are compositions and methods related to multivalent
compositions targeted to cells and tissues. The disclosed targeting
is useful for treatment of cancer and other diseases and
disorders.
Inventors: |
Agemy; Lilach; (La Jolla,
CA) ; Friedmann-Morvinski; Dinorah; (San Diego,
CA) ; Kotamraju; Venkata Ramana; (La Jolla, US)
; Ruoslahti; Erkki; (La Jolla, US) ; Sugahara;
Kazuki; (La Jolla, US) ; Verma; Inder; (La
Jolla, US) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanford Burnham Prebys Medical Discovery Institute
The Salk Institute for Biological Studies |
La Jolla
La Jolla |
CA
CA |
US
US |
|
|
Family ID: |
44120964 |
Appl. No.: |
15/376321 |
Filed: |
December 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13083176 |
Apr 8, 2011 |
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15376321 |
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61322207 |
Apr 8, 2010 |
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61376856 |
Aug 25, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; A61K 38/04 20130101; A61K 47/55 20170801; A61K
47/66 20170801; A61K 47/6923 20170801; B82Y 5/00 20130101 |
International
Class: |
A61K 38/04 20060101
A61K038/04; B82Y 5/00 20060101 B82Y005/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
Nos. 5 P30 CA 30199-28 and P01 CA 104898-01 awarded by the National
Institutes of Health (NIH), and DOD/USAMRAA Grant No. PC 093283
awarded by the Department of Defense (DOD). The government has
certain rights in this invention.
Claims
1. A composition comprising a surface molecule, one or more homing
molecules, and a plurality of membrane perturbing molecules,
wherein the one or more homing molecules selectively home to tumor
vasculature, and further wherein: (i) the one or more of the homing
molecules comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative derivative thereof, the amino acid sequence CRKDKC
(SEQ ID NO:2) or a conservative derivative thereof, or any
combination thereof; (ii) the membrane perturbing molecules
comprise the amino acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID
NO:3) or a conservative variant thereof, (KLAKLAK).sub.2 (SEQ ID
NO:3) or a conservative variant thereof, (KLAKKLA).sub.2 (SEQ ID
NO:5) or a conservative variant thereof, (KAAKKAA).sub.2 (SEQ ID
NO:6) or a conservative variant thereof, or (KLGKKLG).sub.3 (SEQ ID
NO:7) or a conservative variant thereof, or any combination
thereof.
2-9. (canceled)
10. The composition of claim 1, wherein one or more of the membrane
perturbing molecules are conjugated to one or more of the homing
molecules.
11-12. (canceled)
13. The composition of claim 1, wherein the homing molecules, the
membrane perturbing molecules, or both are conjugated with the
surface molecule.
14-21. (canceled)
22. The composition of claim 1, wherein one or more of the membrane
perturbing molecules are covalently coupled to the surface
molecule.
23-24. (canceled)
25. The composition of claim 13, wherein one or more of the
conjugated homing molecules are indirectly conjugated to the
surface molecule via a linker, one or more of the conjugated
membrane perturbing molecules are indirectly conjugated to the
surface molecule via a linker, or both.
26-27. (canceled)
28. The composition of claim 1, wherein the composition further
comprises one or more internalization elements.
29. The composition of claim 28, wherein one or more of the homing
molecules and/or one or more of the membrane perturbing molecules
comprise one or more of the internalization elements.
30-31. (canceled)
32. The composition of claim 1, wherein the composition further
comprises one or more tissue penetration elements.
33. The composition of claim 32, wherein one or more of the tissue
penetration elements are comprised in an internalization
element.
34. The composition of claim 32, wherein the tissue penetration
element is a CendR element.
35. The composition of claim 1, wherein the composition binds
inside tumor blood vessels, is internalized in cells, penetrates
tissue, or reduces tumor growth.
36-38. (canceled)
39. The composition of claim 1, wherein the surface molecule
comprises a nanoparticle, optionally an iron oxide nanoparticle or
an albumin nanoparticle, a nanoworm, optionally an iron oxide
nanoworm, a liposome, a micelle, a phospholipid, a polymer, a
microparticle, or a fluorocarbon microbubble.
40-62. (canceled)
63. The composition of claim 1, further comprising a moiety
selected from the group consisting of an anti-angiogenic agent, a
pro-angiogenic agent, a cancer chemotherapeutic agent, a cytotoxic
agent, an anti-inflammatory agent, an anti-arthritic agent, a
polypeptide, a nucleic acid molecule, a small molecule, an image
contrast agent, a fluorophore, fluorescein, rhodamine, a
radionuclide. indium-111, technetium-99, carbon-11, and
carbon-13.
64. (canceled)
65. The composition of claim 63, wherein at least one of the
moieties is a therapeutic agent.
66. The composition of claim 65, wherein the therapeutic agent is
selected from the group consisting of Abraxane, paclitaxel, and
taxol.
67-69. (canceled)
70. The composition of claims 63, wherein at least one of the
moieties is a detectable agent, optionally FAM.
71. (canceled)
72. The composition of claim 1, wherein: (i) one or more of the
homing molecules comprise the amino acid sequence CGKRK (SEQ ID
NO:1) or a conservative derivative thereof; (ii) one or more of the
membrane perturbing molecules comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3); (iv) one or more of the
conjugated homing molecules are indirectly conjugated to the
surface molecule via a linker; and (v) one or more of the
conjugated membrane perturbing molecules are indirectly conjugated
to the surface molecule via a linker.
73. The composition of claim 72, wherein at least one of the
linkers comprises polyethylene glycol.
74. A method comprising administering to a subject the composition
of claim 1, wherein the composition selectively homes to tumor
vasculature in the subject, wherein the composition is internalized
into cells at the site of the tumor vasculature.
75-81. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/083,176, filed Apr. 8, 2011 (now
abandoned), which itself claims benefit of U.S. Provisional
Application No. 61/322,207, filed Apr. 8, 2010, and U.S.
Provisional Application No. 61/376,856, filed Aug. 25, 2010.
application Ser. No. 13/083, 176, filed Apr. 8, 2011, Application
No. 61/322,207, filed Apr. 8, 2010, and Application No. 61/376,856,
filed Aug. 25, 2010, are hereby incorporated herein by reference in
their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of
molecular medicine, and, more specifically, to compositions that
home to targeted cells and tissue.
BACKGROUND OF THE INVENTION
[0004] A major hurdle to advances in treating cancer is the
relative lack of agents that can selectively target the cancer
while sparing normal tissue. For example, radiation therapy and
surgery, which generally are localized treatments, can cause
substantial damage to normal tissue in the treatment field,
resulting in scarring and loss of normal tissue. Chemotherapy, in
comparison, which generally is administered systemically, can cause
substantial damage to organs such as the bone marrow, mucosae, skin
and small intestine, which undergo rapid cell turnover and
continuous cell division. As a result, undesirable side effects
such as nausea, loss of hair and drop in blood cell count often
occur when a cancer patient is treated intravenously with a
chemotherapeutic drug. Such undesirable side effects can limit the
amount of a drug that can be safely administered, thereby hampering
survival rate and impacting the quality of patient life.
[0005] Nanomedicine is an emerging field that uses nanoparticles to
facilitate the diagnosis and treatment of diseases. Notable early
successes in the clinic include the use of superparamagnetic
nanoparticles as a contrast agent in MRI and nanoparticle-based
treatment systems (Desai 2006; Weissleder 1995). The first
generation of nanoparticles used in tumor treatments rely on
"leakiness" of tumor vessels for preferential accumulation in
tumors; however, this enhanced permeability and retention (EPR) is
not a constant feature of tumor vessels (Sinek 2004) and even when
present, still leaves the nanoparticles to negotiate the high
interstitial fluid pressure in tumors (Sinek 2004; Boucher 1990).
An attractive alternative is to target nanoparticles to specific
molecular receptors in the blood vessels because they are readily
available for binding from the blood stream and because tumor
vessels express a wealth of molecules that are not significantly
expressed in the vessels of normal tissues (Hoffman 2003; Oh 2004;
Ruoslahti 2002).
[0006] Glioblastomas multiforme (GBM) are the most common and
lethal form of intracranial tumors. They account for approximately
70% of the 22,500 new cases of malignant primary brain tumors that
are diagnosed in adults in the United States each year. Although
relatively uncommon, malignant gliomas are associated with
disproportionately high morbidity and mortality (median survival is
only 12 to 15 months). Malignant gliomas are among the most
vascular of human tumors. However, gliomas are among the most
difficult cancers to treat. Their location in the brain makes them
inaccessible to numerous drugs and therapeutic compositions.
Treatments that can effectively target gliomas are needed.
[0007] Specific targeting of nanoparticles to tumors has been
accomplished in various experimental systems (DeNardo 2005; Akerman
2002; Cai 2006), but the efficiency of delivery is generally low.
In nature, amplified homing is an important mechanism ensuring
sufficient platelet accumulation at sites of vascular injury. It
involves target binding, activation, platelet-platelet binding, and
formation of a blood clot.
BRIEF SUMMARY OF THE INVENTION
[0008] Disclosed are compositions and methods useful for delivering
significant amounts of compounds of interest to targeted cells and
tissues. The disclosed compositions and methods are useful, for
example, to deliver to targeted cells and tissues an effective
amount of compounds that are excessively toxic. For example,
disclosed are compositions comprising a surface molecule, one or
more homing molecules, and a plurality of cargo molecules. The
cargo molecules can be, for example, excessively toxic molecules.
The cargo molecules can be, for example, membrane perturbing
molecules. As another example, disclosed are compositions
comprising a surface molecule, one or more homing molecules, and a
plurality of membrane perturbing molecules. Also disclosed are
methods comprising, for example, administering to a subject the
disclosed compositions.
[0009] The homing molecules can home to targets of interest, such
as cells and tissues of interest. For example, the homing molecules
can home to tumor vasculature. The homing molecules can selectively
home to targets of interest, such as cells and tissues of interest.
For example, the homing molecules can selectively homes to tumor
vasculature. The composition can home to one or more of the sites
to be targeted. The composition can be internalized in cells. The
composition can penetrate tissue. The composition can be
internalized into cells at the targeted site. The composition can
penetrate tissue at the targeted site. The composition can, for
example be internalized into cancer cells. The composition can, for
example, penetrate tumor tissue. The composition can, for example,
bind inside tumor blood vessels.
[0010] In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative derivative thereof, the amino acid sequence CRKDKC
(SEQ ID NO:2) or a conservative derivative thereof, or a
combination. In some forms, one or more of the homing molecule can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative variant thereof. In some forms, one or more of the
homing molecules can comprise the amino acid sequence CGKRK (SEQ ID
NO:1). In some forms, one or more of the membrane perturbing
molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative variant
thereof, (KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative variant
thereof, (KLAKKLA).sub.2 (SEQ ID NO:5) or a conservative variant
thereof, (KAAKKAA).sub.2 (SEQ ID NO:6) or a conservative variant
thereof, (KLGKKLG).sub.3 (SEQ ID NO:7) or a conservative variant
thereof, or a combination. In some forms, one or more of the
membrane perturbing molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3), (KLAKLAK).sub.2 (SEQ ID NO:3),
(KLAKKLA).sub.2 (SEQ ID NO:5), (KAAKKAA).sub.2 (SEQ ID NO:6),
(KLGKKLG).sub.3 (SEQ ID NO:7), or a combination. In some forms, one
or more of the membrane perturbing molecules can comprise the amino
acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative
variant thereof. In some forms, one or more of the membrane
perturbing molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3).
[0011] In some forms, the composition comprises a plurality of
surface molecules, a plurality of homing molecules and a plurality
of cargo molecules. In some forms, the composition comprises one or
more surface molecules, a plurality of homing molecules and a
plurality of cargo molecules. In some forms, the composition
comprises a plurality of surface molecules, one or more homing
molecules and a plurality of cargo molecules. In some forms, the
composition comprises a plurality of surface molecules, a plurality
of homing molecules and one or more cargo molecules. In some forms,
the composition comprises one or more surface molecules, one or
more homing molecules and a plurality of cargo molecules. In some
forms, the composition comprises one or more surface molecules, a
plurality of homing molecules and one or more cargo molecules. In
some forms, the composition comprises a plurality of surface
molecules, one or more homing molecules and one or more cargo
molecules.
[0012] In some forms, the composition comprises a surface molecule,
a plurality of homing molecules and a plurality of cargo molecules,
wherein one or more of the homing molecules and one or more of the
cargo molecules are associated with the surface molecule. In some
forms, the composition comprises a surface molecule, a plurality of
homing molecules and a plurality of cargo molecules, wherein a
plurality of the plurality of homing molecules and a plurality of
the plurality of cargo molecules are associated with the surface
molecule. In some forms, the composition comprises a surface
molecule, a plurality of homing molecules and a plurality of cargo
molecules, wherein the homing molecules and the cargo molecules are
associated with the surface molecule.
[0013] In some forms, the composition comprises a surface molecule,
wherein the surface molecule is multivalent for homing molecules
and cargo molecules. In some forms, the composition comprises a
surface molecule, wherein the surface molecule is multivalent for
homing molecules and comprises one or more cargo molecules. In some
forms, the composition comprises a surface molecule, wherein the
surface molecule is multivalent for cargo molecules and comprises
one or more homing molecules. In some forms, the composition
comprises a surface molecule, wherein the surface molecule is
multivalent for conjugates, wherein one or more of the conjugates
comprise one or more homing molecules and one or more cargo
molecules. In some forms, the composition comprises a surface
molecule, wherein the surface molecule is multivalent for
conjugates, wherein one or more of the conjugates comprise a
plurality of homing molecules and a plurality cargo molecules. In
some forms, the composition comprises a surface molecule, wherein
the surface molecule is multivalent for conjugates, wherein one or
more of the conjugates comprise a homing molecule and a cargo
molecule. In some forms, the composition comprises a surface
molecule, wherein the surface molecule is multivalent for
conjugates, wherein each of the conjugates comprises a plurality of
homing molecules and a plurality cargo molecules. In some forms,
the composition comprises a surface molecule, wherein the surface
molecule is multivalent for conjugates, wherein each of the
conjugates comprises a homing molecule and a cargo molecule.
[0014] In some forms, the composition comprises a surface molecule,
wherein the surface molecule comprises one or more conjugates,
wherein one or more of the conjugates comprise one or more homing
molecules and one or more cargo molecules. In some forms, the
composition comprises a surface molecule, wherein the surface
molecule comprises one or more conjugates, wherein one or more of
the conjugates comprise a plurality of homing molecules and a
plurality cargo molecules. In some forms, the composition comprises
a surface molecule, wherein the surface molecule comprises one or
more conjugates, wherein one or more of the conjugates comprise a
homing molecule and a cargo molecule. In some forms, the
composition comprises a surface molecule, wherein the surface
molecule comprises one or more conjugates, wherein each of the
conjugates comprises a plurality of homing molecules and a
plurality cargo molecules. In some forms, the composition comprises
a surface molecule, wherein the surface molecule comprises one or
more conjugates, wherein each of the conjugates comprises a homing
molecule and a cargo molecule.
[0015] In some forms, one or more of the membrane perturbing
molecules can be conjugated to one or more of the homing molecules.
In some forms, one or more of the conjugated membrane perturbing
molecules and homing molecules can be covalently coupled. In some
forms, one or more of the covalently coupled membrane perturbing
molecules and homing molecules can comprise fusion peptides. In
some forms, the homing molecules can be conjugated with the surface
molecule. In some forms, one or more of the conjugated homing
molecules can be directly conjugated to the surface molecule. In
some forms, one or more of the conjugated homing molecules can be
indirectly conjugated to the surface molecule. In some forms, one
or more of the homing molecules can be covalently coupled to the
surface molecule. In some forms, one or more of the covalently
coupled homing molecules can be directly covalently coupled to the
surface molecule. In some forms, one or more of the covalently
coupled homing molecules can be indirectly covalently coupled to
the surface molecule. In some forms, the membrane perturbing
molecules can be conjugated with the surface molecule. In some
forms, one or more of the conjugated membrane perturbing molecules
are directly conjugated to the surface molecule. In some forms, one
or more of the conjugated membrane perturbing molecules can be
indirectly conjugated to the surface molecule. In some forms, one
or more of the membrane perturbing molecules can be covalently
coupled to the surface molecule. In some forms, one or more of the
covalently coupled membrane perturbing molecules can be directly
covalently coupled to the surface molecule. In some forms, one or
more of the covalently coupled membrane perturbing molecules can be
indirectly covalently coupled to the surface molecule.
[0016] In some forms, the composition can further comprise one or
more internalization elements. In some forms, one or more of the
homing molecules can comprise one or more of the internalization
elements. In some forms, one or more of the membrane perturbing
molecules can comprise one or more of the internalization elements.
In some forms, the surface molecule can comprise one or more of the
internalization elements not comprised in either the homing
molecules or the membrane perturbing molecules. In some forms, the
composition can further comprise one or more tissue penetration
elements. In some forms, one or more of the tissue penetration
elements can be comprised in an internalization element. In some
forms, the tissue penetration element can be a CendR element.
[0017] In some forms, the surface molecule can comprise a
nanoparticle. In some forms, the surface molecule can comprise a
nanoworm. In some forms, the surface molecule can comprise an iron
oxide nanoworm. In some forms, the surface molecule can comprise an
iron oxide nanoparticle. In some forms, the surface molecule can
comprise an albumin nanoparticle. In some forms, the surface
molecule can comprise a liposome. In some forms, the surface
molecule can comprise a micelle. In some forms, the surface
molecule comprises a phospholipid. In some forms, the surface
molecule comprises a polymer. In some forms, the surface molecule
can comprise a microparticle. In some forms, the surface molecule
can comprise a fluorocarbon microbubble.
[0018] In some forms, the composition can comprise at least 100
homing molecules. In some forms, the composition can comprise at
least 1000 homing molecules. In some forms, the composition can
comprise at least 10,000 homing molecules. In some forms, the
composition can comprise at least 100 membrane perturbing
molecules. In some forms, the composition can comprise at least
1000 membrane perturbing molecules. In some forms, the composition
can comprise at least 10,000 membrane perturbing molecules.
[0019] In some forms, one or more of the homing molecules can be
modified homing molecules. In some forms, one or more of the homing
molecules can comprise a methylated homing molecule. In some forms,
one or more of the methylated homing molecules can comprise a
methylated amino acid segment. In some forms, one or more of the
membrane perturbing molecules can be modified membrane perturbing
molecules. In some forms, one or more of the membrane perturbing
molecules comprise a methylated membrane perturbing molecule. In
some forms, one or more of the methylated membrane perturbing
molecules comprise a methylated amino acid segment. In some forms,
the amino acid sequence is N- or C-methylated in at least one
position.
[0020] In some forms, the composition can further comprise one or
more moieties. In some forms, the moieties can be independently
selected from the group consisting of an anti-angiogenic agent, a
pro-angiogenic agent, a cancer chemotherapeutic agent, a cytotoxic
agent, an anti-inflammatory agent, an anti-arthritic agent, a
polypeptide, a nucleic acid molecule, a small molecule, an image
contrast agent, a fluorophore, fluorescein, rhodamine, a
radionuclide, indium-111, technetium-99, carbon-11, and carbon-13.
In some forms, at least one of the moieties can be a therapeutic
agent. In some forms, the therapeutic agent can be iRGD, RGD,
Abraxane, paclitaxel, taxol, or a combination. In some forms, at
least one of the moieties can be a detectable agent. In some forms,
the detectable agent can be FAM.
[0021] In some forms, the composition can have a therapeutic
effect. In some forms, the composition can reduce tumor growth. In
some forms, the therapeutic effect can be a slowing in the increase
of or a reduction of tumor burden. In some forms, the therapeutic
effect can be a slowing of the increase of or reduction of tumor
size. In some forms, the subject can have one or more sites
targeted, wherein the composition can home to one or more of the
sites targeted. In some forms, the subject can have a tumor,
wherein the composition can have a therapeutic effect on the
tumor.
[0022] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0024] FIG. 1 is a schematic diagram of an apoptotic cell.
[0025] FIGS. 2A, 2B and 2C show cytotoxicity of
.sub.D(KLAKLAK).sub.2CGKRK peptide in cell lines. FIGS. 2A and 2B
show the cytotoxicity of .sub.D(KLAKLAK).sub.2CGKRK peptide in
human umbilical vein endothelial cells (HUVEC) (A) and T3 (B)
cells. FIG. 2C shows the cytotoxicity of .sub.D(KLAKLAK).sub.2CGKRK
in U87 cells Cultured cells were treated CGKRK, or
.sub.D(KLAKLAK).sub.2, or .sub.D(KLAKLAK).sub.2CGKRK peptide. The
cells were incubated with peptide for 24 hrs and cell death was
quantified by MTT assays (n=3). Statistical analyses were performed
with Student's t-test. Error bars, s.e.m.
[0026] FIGS. 3A, 3B and 3C show cytotoxicity of
.sub.D(KLAKLAK).sub.2CGKRK conjugated with NW HUVEC cells. Cultured
HUVEC cells were treated with non-targeted .sub.D(KLAKLAK).sub.2
conjugated NW (D(KLAKLAK)2), CREKA conjugated NW (CREKA), CGKRK
conjugated NW (CGKRK), or CGKRK-.sub.D(KLAKLAK).sub.2 conjugated NW
(D(KLAKLAK)2-CGKRK). The cells were incubated with NW for 48 hrs
without washing (A and C) or the NW were washed after 20 min (B)
and cell death was quantified by MTT assays (n=3). FIG. 3A used
rapidly proliferating HUVEC cells while FIG. 3C used synchronized
HUVEC cells. Statistical analyses were performed with Student's
t-test. Error bars, s.e.m.
[0027] FIG. 4 shows cytotoxicity of CGKRK-.sub.D(KLAKLAK).sub.2
conjugated with NW in T3 cells. Cultured T3 cells were treated with
non-targeted .sub.D(KLAKLAK).sub.2 conjugated NW (D(KLAKLAK)2),
CREKA conjugated NW (CREKA), CGKRK conjugated NW (CGKRK), or
CGKRK-.sub.D(KLAKLAK).sub.2 conjugated with NW (D(KLAKLAK)2-CGKRK).
The cells were incubated with NW for 48 hrs and cell death was
quantified by MTT assay.
[0028] FIGS. 5A and 5B show cytotoxicity of
.sub.D(KLAKLAK).sub.2CGKRK conjugated with NW in U87 cells.
Cultured U87 cells were treated with non-targeted
.sub.D(KLAKLAK).sub.2 conjugated NW (shown as KLAKLAK-NW on the
graph), KAKEC (SEQ ID NO:135) conjugated NW (KAKEC-NW), CGKRK
conjugated NW (CGKRK-NW), or CGKRK-.sub.D(KLAKLAK).sub.2 conjugated
with NW (CIMERA-NW). The cells were incubated with NW for 24 or 48
hrs and cell death was quantified by MTT assays. These results are
almost the same results seen with U251 which had 50-60% cell
viability.
[0029] FIG. 6 shows the IC50 of .sub.D(KLAKLAK).sub.2CGKRK peptide
versus peptide on nanoworms. NW coated with
.sub.D(KLAKLAK).sub.2CGKRK via a 5-kDa PEG-linker were cleaved from
the particles using DTT and the amount of peptide present on the
particle was calculated to compare the amount of free peptide
versus the peptide coated nanoparticle IC50 values.
[0030] FIG. 7 shows .sub.D(KLAKLAK).sub.2CGKRK conjugated with NW
induced apoptosis in HUVEC cells. HUVEC cells were left untreated
(Control) or treated for 24, 48 and 72 hrs with an irrelevant
peptide-NW (CREKA-NW; SEQ ID NO:92) or the
.sub.D(KLAKLAK).sub.2CGKRK-NW. Cells were incubated with Annexin
V-PE in a buffer containing 7-Amino-actinomycin (7-AAD) and
analyzed by flow cytometry. The percentage of Annexin V positive
cells (apoptotic cells plus end stage apoptosis or already dead
cells) is indicated in each graph.
[0031] FIG. 8 shows .sub.D(KLAKLAK).sub.2CGKRK conjugated with NW
induced apoptosis in T3 cells. T3 cells (tumor endothelial cells)
were left untreated (Control) or treated for 24 and 48 hrs with an
irrelevant peptide-NW (CREKA-NW; SEQ ID NO:92) or the
.sub.D(KLAKLAK).sub.2CGKRK-NW. Cells were incubated with Annexin
V-PE in a buffer containing 7-Amino-actinomycin (7-AAD) and
analyzed by flow cytometry. The percentage of Annexin V positive
cells (apoptotic cells plus end stage apoptosis or already dead
cells) is indicated in each graph.
[0032] FIG. 9 shows .sub.D(KLAKLAK).sub.2CGKRK conjugated with NW
inhibits HUVEC capillary-like tube formation in vitro. Primary
HUVECs were plated on growth factor reduced matrigel in 5% FCS
medium alone (control), or containing CGKRK-NW (SEQ ID NO:92) (10
microg/ml), or containing .sub.D(KLAKLAK).sub.2CGKRK-NW (5 and 10
microg/ml). The formation of networks of capillary-like structures
was viewed by phase contrast-microscopy at 40.times. magnification
24 h after plating.
[0033] FIG. 10 shows caspase activity by HUVEC cells treated with
.sub.D(KLAKLAK).sub.2CGKRK-NW. Caspase-3 activity was determined in
HUVEC cells 24 h after treatment with 3 or 10 microgram
.sub.D(KLAKLAK).sub.2CGKRK-NW using a caspase-Glo 3/7 assay kit.
Two hours after reagent was added luminescence was recorded on
luminometer.
[0034] FIG. 11 is a diagram of the glioblastomas multiforme (GBM)
treatment with CGKRK-.sub.D(KLAKLAK).sub.2-NW nanoworms (EXP NUMBER
1). Mice bearing RAS-sip53 induced brain tumors (three weeks
post-injection) were intravenously injected with NW coated with
peptides through a 5-kDa polyethylene glycol spacer. The particles
were administered every other day for 14 days (5 mg iron/kg/day,
total cumulative dose 35 mg/kg). Survival was monitored over time
(n=3 per group).
[0035] FIG. 12 shows GBM treatment with
CGKRK-.sub.D(KLAKLAK).sub.2-NW nanoworms (EXP NUMBER 1). Mice
bearing RAS-sip53 induced brain tumors (three weeks post-injection)
were intravenously injected with NW coated with peptides through a
5-kDa polyethylene glycol spacer. The particles were administered
every other day for 14 days (5 mg iron/kg/day, total cumulative
dose 35 mg/kg). Survival was monitored over time (n=3 per
group).
[0036] FIGS. 13A and 13B show GBM treatment with
CGKRK-.sub.D(KLAKLAK).sub.2-NW nanoworms (EXP NUMBER 2). Mice
bearing RAS-sip53 induced brain tumors (injection to the right
hippocampus) were intravenously injected with NW coated with
peptides through a 5-kDa polyethylene glycol spacer. The particles
alone or co-injection with iRGD were administered once a week for 6
weeks (one weeks post-viral injection) or every other day for two
weeks and a half weeks (three weeks post-viral injection). All mice
were monitored for luciferase signal using the IVIS system (the
lentivector contains the luciferase reporter), only one
representative mouse from the indicated groups is shown in the
figure. Survival of the mice is being currently recorded (n=3 per
group).
[0037] FIG. 14 shows ALT (L-Alanine-2-Oxoglutarate
Aminotransferase) levels in mice pre and post-nanoworm treatment.
Mice were bled one day before starting the treatment and one day
following the two and a half treatment course. For the groups of
mice injected every other day another blood collection was
performed two weeks after the last day of treatment. The levels of
ALT were tested in the serum of all the mice. Normal values go from
10-40 U/L.
[0038] FIGS. 15A, 15B and 15C show the GBM treatment with
CGKRK-.sub.D(KLAKLAK).sub.2-NW nanoworms. Panel A shows a schematic
of the experiment. Mice bearing 005 brain tumor cells (10 day
post-injection) were intravenously injected with NW coated with
peptides through a 5-kDa polyethylene glycol spacer. The particles
without and co-injection with iRGD were administered every other
day for 14 days (5 mg iron/kg/day, total cumulative dose 35 mg/kg).
Panel B shows a graph of survival. Survival was monitored over time
(n=3 per group). Panel C shows the results of mice having tumors
induced by injecting 3.times.105 005 cells into the right
hippocampus area. The 005 cell line was derived from a lentivirally
(RAS-sip53) induced brain tumor (3). Ten days after the tumor cell
injection, the mice were intravenously injected with NW. The NWs
were administered every other day for 14 days followed by one week
gap and continued treatment for 14 days. All but 2 control mice
have died of the tumors, whereas all of mice treated with
CGKRK-D[KLAKLAK]2-NW are alive (top line) with no overt signs of a
tumor (n=8 per group). The controls were: no NW (bottom line with
about 15% survival at day 35), D[KLAKLAK]2-NW middle line with
about 40% survival at day 35), and CGKRK-NW (middle line with about
50% survival at day 35).
[0039] FIG. 16 shows the structure of targeted theranostic NW.
Aminated NW were synthesized according to Park et al (4) and
reacted with NHS-PEG(5K)-maleimide. Subsequently, peptides were
coated on the NW through reaction between the maleimide group on
the PEG and a cysteine thiol of the peptide. Coupling through the
side chain of the central cysteine in the D(KLAKLAK)2CGKRK peptide
gives the V-shaped structure depicted in the figure.
[0040] FIG. 17 is a graph of FAM-CGKRK peptide binding to
mitochondria in the presence of unlabeled peptide (left panel) and
a control peptide (right panel). FAM-CGKRK was incubated with
purified mitochondria in the presence of increasing concentrations
of either unlabeled CGKRK or an unrelated peptide (CREKA; SEQ ID
NO:92) as a control.
[0041] FIG. 18 is a graph of phage binding to mitochondria. CGKRK
phage and CREKA (SEQ ID NO:92) phage (as a control) were incubated
with purified mitochondria. Titration of bound phage shows about 80
times more binding of the CGKRK phage than the control. Student's
t-test (c), Error bars, mean.+-.SD; n.s., **p<0.01;
***p<0.001.
[0042] FIG. 19 is a graph of adsorption (A.sub.450 nm) versus
biotin-CGKRK concentration (.mu.M). Binding of increasing amounts
of biotin-labeled CGKRK peptide to immobilized p32 protein was
detected with streptavidin coupled to horseradish peroxidase and
normalized to nonspecific binding in the absence of p32. The
affinity of the peptide for p32 calculated from the binding curves
is also shown. The saturation curve shown is average of three
independent experiments. Error bars, mean.+-.SD.
[0043] FIGS. 20A and 20B are graphs of the percent of inhibition
versus non-labeled peptide added. FIG. 20A shows the results for
biotin labeled CGKRK and FIG. 20B shows the results for biotin
labeled LyP-1 peptide.
[0044] FIG. 21 shows Annexin V positive cells (%) when treated with
various peptide compositions 24, 48 and 72 hours. HUVEC and T3
cells were left untreated (Control) or treated with a concentration
of 10 .mu.g/ml of NWs coated with either a control peptide (CREKA;
SEQ ID NO:92), .sub.D[KLAKLAK].sub.2, or
CGKRK.sub.D[KLAKLAK].sub.2. The cells were stained with Annexin and
analyzed by flow cytometry. The total percentage of
Annexin-positive cells (apoptotic and dead cells) is indicated.
[0045] FIG. 22 shows Annexin V positive cells (%) when treated with
various peptide compositions for 30 minutes (when the particles
were washed away) and the incubation was continued for 72 hrs. The
cells were stained with Annexin and analyzed by flow cytometry. The
total percentage of Annexin-positive cells (apoptotic and dead
cells) is indicated.
[0046] FIG. 23 shows survival (%) versus time (days) for mice
bearing lenti-viral (H-RasV12-sip53) induced brain tumors treated
with .sub.D[KLAKLAK].sub.2-NWs or CGKRK .sub.D[KLAKLAK].sub.2-NWs.
Mice bearing lenti-viral (H-RasV12-sip53) induced brain tumors in
the right hippocampus were intravenously injected with NW coated
with peptides. The particles were administered every other day for
18 days, starting 3 weeks post-viral injection. Survival curve of
the non-treated and treated mice (n=8-10 per group).
[0047] FIG. 24 shows survival (%) versus time (days) for mice
bearing 005 tumor cells treated with .sub.D[KLAKLAK].sub.2-NWs,
CGKRK-NWs, and CGKRK.sub.D[KLAKLAK].sub.2-NWs. Tumors were
developed by transplanting 3.times.10.sup.5 005 cells into the
right hippocampus of NOD-SCID mice. Ten days post-tumor cell
transplantation, the mice were intravenously injected with NWs. The
NWs (5 mg of iron/kg) were administered every other day for 3 weeks
or administered non stop for the same period of time (n=8 per
group). Survival curves of the treated mice are shown.
[0048] FIG. 25 shows survival (%) versus time (days) for mice
bearing 005 tumor cells treated with CGKRK.sub.D[KLAKLAK].sub.2-NWs
with co-administration of cRGD or iRGD. Mice bearing orthotopic 005
tumors implanted 10 days earlier received every other day for 3
weeks intravenous injections of CGKRK.sub.D[KLAKLAK].sub.2-NWs (5
mg of iron/kg) mixed with 4 mmol/kg of cRGD or iRGD. Results for
control mice and mice administered only iRGD are also shown.
Survival curves are shown (n=8-10 per group).
[0049] FIG. 26 shows inhibition of CGKRK peptide binding to p32 by
anti-p32. Biotin-CGKRK at 1 .mu.g/ml was incubated in microtiter
wells coated with purified p32, and the binding was detected with
streptavidin coupled to horseradish peroxidase and normalized to
nonspecific binding in the absence of p32. The anti-32 antibody was
prepared against the full-length p32 protein (Protein Production
and Analysis Facility of the Sanford-Burnham Medical Research
Institute). The experiments were performed in triplicate; one of
two experiments with similar results is shown.
[0050] FIGS. 27A, 27B, and 27C shows that
CGKRK.sub.D[KLAKLAK].sub.2-NW conjugates induce cell death by
apoptosis. HUVEC (A) and T3 (B) cells were left untreated (Control)
or were treated with 10 .mu.g/ml of NWs coated with
CGKRK.sub.D[KLAKLAK].sub.2-NWs for 48 (A) or 72 hours (B). In 27C,
the cells were incubated with the indicated NWs, washed to remove
excess NWs after 30 minutes, and then incubated for 72 hours.
Annexin staining and analysis by flow cytometry were used to
measure apoptosis in the cultures. Representative images are shown
indicating the percentage of Annexin-positive cells (apoptotic and
dead cells).
[0051] FIG. 28 shows toxicology analyses of mice treated with
CGKRK.sub.D[KLAKLAK].sub.2-NWs. Blood L-alanine-2-oxoglutarate
aminotransferase (ALT) levels measured before (Pre-treatment),
after completion of a 3-week treatment course (After treatment),
and after a subsequent 2-week recovery period (2 weeks after
treatment) are shown.
[0052] FIGS. 29A and 29B show toxicology analyses of mice treated
with CGKRK.sub.D[KLAKLAK].sub.2-NWs. Possible active and innate
immune responses against NW was tested by measuring antibody (29A)
and IL-6 levels (29B) in serum form mice treated and collected as
in FIG. 28.
[0053] FIG. 30 shows survival curves of mice bearing intracranial
U87 tumors treated with CGKRK.sub.D[KLAKLAK].sub.2-NWs. Tumors were
induced by injecting 5.times.10.sup.5 GFP-expressing U87 cells into
the right hippocampus of mice. Treatment with intravenous
injections of CGKRK-.sub.D[KLAKLAK].sub.2-NWs and control NWs was
started 10 days after the tumor cell injection and continued every
other day for 3 weeks (n=5 per group).
DETAILED DESCRIPTION OF THE INVENTION
[0054] The disclosed methods and compositions can be understood
more readily by reference to the following detailed description of
particular embodiments and the Example included therein and to the
Figures and their previous and following description.
[0055] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that they are not limited to specific synthetic methods
or specific recombinant biotechnology methods unless otherwise
specified, or to particular reagents unless otherwise specified, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to be limiting.
DEFINITIONS
[0056] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a pharmaceutical carrier" includes mixtures of two or
more such carriers, and the like.
[0057] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15. It is
also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0058] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0059] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
[0060] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon.
[0061] It is to be understood that the disclosed method and
compositions are not limited to specific synthetic methods,
specific analytical techniques, or to particular reagents unless
otherwise specified, and, as such, may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting.
MATERIALS
[0062] Disclosed are the components to be used to prepare the
disclosed compositions as well as the compositions themselves to be
used within the methods disclosed herein. These and other materials
are disclosed herein, and it is understood that when combinations,
subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual
and collective combinations and permutation of these compounds may
not be explicitly disclosed, each is specifically contemplated and
described herein. For example, if a particular peptide is disclosed
and discussed and a number of modifications that can be made to a
number of molecules including the peptide are discussed,
specifically contemplated is each and every combination and
permutation of the peptides and the modifications that are possible
unless specifically indicated to the contrary. Thus, if a class of
molecules A, B, and C are disclosed as well as a class of molecules
D, E, and F and an example of a combination molecule, A-D is
disclosed, then even if each is not individually recited each is
individually and collectively contemplated meaning combinations,
A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered
disclosed. Likewise, any subset or combination of these is also
disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E
would be considered disclosed. This concept applies to all aspects
of this application including, but not limited to, steps in methods
of making and using the disclosed compositions. Thus, if there are
a variety of additional steps that can be performed it is
understood that each of these additional steps can be performed
with any specific embodiment or combination of embodiments of the
disclosed methods.
[0063] Disclosed are compositions useful for delivering significant
amounts of compounds of interest to targeted cells and tissues. The
disclosed compositions are useful, for example, to deliver to
targeted cells and tissues an effective amount of compounds that
are excessively toxic. For example, disclosed are compositions
comprising a surface molecule, one or more homing molecules, and a
plurality of cargo molecules. The cargo molecules can be, for
example, excessively toxic molecules. The cargo molecules can be,
for example, membrane perturbing molecules. As another example,
disclosed are compositions comprising a surface molecule, one or
more homing molecules, and a plurality of membrane perturbing
molecules. As used herein, excessively toxic compounds are
compounds that too toxic when administered to a subject in
unconjugated forms in what would be a therapeutically effective
amount but for the toxicity.
[0064] The homing molecules can home to targets of interest, such
as cells and tissues of interest. For example, the homing molecules
can home to tumor vasculature. The homing molecules can selectively
home to targets of interest, such as cells and tissues of interest.
For example, the homing molecules can selectively home to tumor
vasculature. The composition can home to one or more of the sites
to be targeted. The composition can be internalized in cells. The
composition can penetrate tissue. The composition can be
internalized into cells at the targeted site. The composition can
penetrate tissue at the targeted site. The composition can, for
example be internalized into cancer cells. The composition can, for
example, penetrate tumor tissue. The composition can, for example,
bind inside tumor blood vessels.
[0065] In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative derivative thereof, the amino acid sequence CRKDKC
(SEQ ID NO:2) or a conservative derivative thereof, or a
combination. In some forms, one or more of the homing molecule can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative variant thereof. In some forms, one or more of the
homing molecules can comprise the amino acid sequence CGKRK (SEQ ID
NO:1). In some forms, one or more of the membrane perturbing
molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative variant
thereof, (KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative variant
thereof, (KLAKKLA).sub.2 (SEQ ID NO:5) or a conservative variant
thereof, (KAAKKAA).sub.2 (SEQ ID NO:6) or a conservative variant
thereof, (KLGKKLG).sub.3 (SEQ ID NO:7) or a conservative variant
thereof, or a combination. In some forms, one or more of the
membrane perturbing molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3), (KLAKLAK).sub.2 (SEQ ID NO:3),
(KLAKKLA).sub.2 (SEQ ID NO:5), (KAAKKAA).sub.2 (SEQ ID NO:6),
(KLGKKLG).sub.3 (SEQ ID NO:7), or a combination. In some forms, one
or more of the membrane perturbing molecules can comprise the amino
acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative
variant thereof. In some forms, one or more of the membrane
perturbing molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3).
[0066] In some forms, the composition can comprise a plurality of
surface molecules, a plurality of homing molecules and a plurality
of cargo molecules. In some forms, the composition can comprise one
or more surface molecules, a plurality of homing molecules and a
plurality of cargo molecules. In some forms, the composition can
comprise a plurality of surface molecules, one or more homing
molecules and a plurality of cargo molecules. In some forms, the
composition can comprise a plurality of surface molecules, a
plurality of homing molecules and one or more cargo molecules. In
some forms, the composition can comprise one or more surface
molecules, one or more homing molecules and a plurality of cargo
molecules. In some forms, the composition can comprise one or more
surface molecules, a plurality of homing molecules and one or more
cargo molecules. In some forms, the composition comprises a
plurality of surface molecules, one or more homing molecules and
one or more cargo molecules.
[0067] In some forms, the composition can comprise a surface
molecule, a plurality of homing molecules and a plurality of cargo
molecules, wherein one or more of the homing molecules and one or
more of the cargo molecules are associated with the surface
molecule. In some forms, the composition can comprise a surface
molecule, a plurality of homing molecules and a plurality of cargo
molecules, wherein a plurality of the plurality of homing molecules
and a plurality of the plurality of cargo molecules are associated
with the surface molecule. In some forms, the composition can
comprise a surface molecule, a plurality of homing molecules and a
plurality of cargo molecules, wherein the homing molecules and the
cargo molecules are associated with the surface molecule.
[0068] In some forms, the composition can comprise a surface
molecule, wherein the surface molecule is multivalent for homing
molecules and cargo molecules. In some forms, the composition can
comprise a surface molecule, wherein the surface molecule is
multivalent for homing molecules and comprises one or more cargo
molecules. In some forms, the composition can comprise a surface
molecule, wherein the surface molecule is multivalent for cargo
molecules and comprises one or more homing molecules. In some
forms, the composition can comprise a surface molecule, wherein the
surface molecule is multivalent for conjugates, wherein one or more
of the conjugates comprise one or more homing molecules and one or
more cargo molecules. In some forms, the composition can comprise a
surface molecule, wherein the surface molecule is multivalent for
conjugates, wherein one or more of the conjugates comprise a
plurality of homing molecules and a plurality cargo molecules. In
some forms, the composition can comprise a surface molecule,
wherein the surface molecule is multivalent for conjugates, wherein
one or more of the conjugates comprise a homing molecule and a
cargo molecule. In some forms, the composition can comprise a
surface molecule, wherein the surface molecule is multivalent for
conjugates, wherein each of the conjugates comprises a plurality of
homing molecules and a plurality cargo molecules. In some forms,
the composition can comprise a surface molecule, wherein the
surface molecule is multivalent for conjugates, wherein each of the
conjugates comprises a homing molecule and a cargo molecule. As
used herein, a component that is stated to be "multivalent for" one
or more other components refers to a component that has a plurality
of the other components associated with, conjugated to and/or
covalent coupled to the first component.
[0069] In some forms, the composition can comprise a surface
molecule, wherein the surface molecule comprises one or more
conjugates, wherein one or more of the conjugates comprise one or
more homing molecules and one or more cargo molecules. In some
forms, the composition can comprise a surface molecule, wherein the
surface molecule comprises one or more conjugates, wherein one or
more of the conjugates comprise a plurality of homing molecules and
a plurality cargo molecules. In some forms, the composition can
comprise a surface molecule, wherein the surface molecule comprises
one or more conjugates, wherein one or more of the conjugates
comprise a homing molecule and a cargo molecule. In some forms, the
composition can comprise a surface molecule, wherein the surface
molecule comprises one or more conjugates, wherein each of the
conjugates comprises a plurality of homing molecules and a
plurality cargo molecules. In some forms, the composition can
comprise a surface molecule, wherein the surface molecule comprises
one or more conjugates, wherein each of the conjugates comprises a
homing molecule and a cargo molecule.
[0070] In some forms, one or more of the membrane perturbing
molecules can be conjugated to one or more of the homing molecules.
In some forms, one or more of the conjugated membrane perturbing
molecules and homing molecules can be covalently coupled. In some
forms, one or more of the covalently coupled membrane perturbing
molecules and homing molecules can comprise fusion peptides. In
some forms, the homing molecules can be conjugated with the surface
molecule. In some forms, one or more of the conjugated homing
molecules can be directly conjugated to the surface molecule. In
some forms, one or more of the conjugated homing molecules can be
indirectly conjugated to the surface molecule. In some forms, one
or more of the homing molecules can be covalently coupled to the
surface molecule. In some forms, one or more of the covalently
coupled homing molecules can be directly covalently coupled to the
surface molecule. In some forms, one or more of the covalently
coupled homing molecules can be indirectly covalently coupled to
the surface molecule. In some forms, the membrane perturbing
molecules can be conjugated with the surface molecule. In some
forms, one or more of the conjugated membrane perturbing molecules
are directly conjugated to the surface molecule. In some forms, one
or more of the conjugated membrane perturbing molecules can be
indirectly conjugated to the surface molecule. In some forms, one
or more of the membrane perturbing molecules can be covalently
coupled to the surface molecule. In some forms, one or more of the
covalently coupled membrane perturbing molecules can be directly
covalently coupled to the surface molecule. In some forms, one or
more of the covalently coupled membrane perturbing molecules can be
indirectly covalently coupled to the surface molecule.
[0071] In some forms, the composition can further comprise one or
more internalization elements. In some forms, one or more of the
homing molecules can comprise one or more of the internalization
elements. In some forms, one or more of the membrane perturbing
molecules can comprise one or more of the internalization elements.
In some forms, the surface molecule can comprise one or more of the
internalization elements not comprised in either the homing
molecules or the membrane perturbing molecules. In some forms, the
composition can further comprise one or more tissue penetration
elements. In some forms, one or more of the tissue penetration
elements can be comprised in an internalization element. In some
forms, the tissue penetration element can be a CendR element.
[0072] In some forms, the surface molecule can comprise a
nanoparticle. In some forms, the surface molecule can comprise a
nanoworm. In some forms, the surface molecule can comprise an iron
oxide nanoworm. In some forms, the surface molecule can comprise an
iron oxide nanoparticle. In some forms, the surface molecule can
comprise an albumin nanoparticle. In some forms, the surface
molecule can comprise a liposome. In some forms, the surface
molecule can comprise a micelle. In some forms, the surface
molecule comprises a phospholipid. In some forms, the surface
molecule comprises a polymer. In some forms, the surface molecule
can comprise a microparticle. In some forms, the surface molecule
can comprise a fluorocarbon microbubble.
[0073] In some forms, the composition can comprise at least 100
homing molecules. In some forms, the composition can comprise at
least 1000 homing molecules. In some forms, the composition can
comprise at least 10,000 homing molecules. In some forms, the
composition can comprise at least 100 membrane perturbing
molecules. In some forms, the composition can comprise at least
1000 membrane perturbing molecules. In some forms, the composition
can comprise at least 10,000 membrane perturbing molecules.
[0074] In some forms, one or more of the homing molecules can be
modified homing molecules. In some forms, one or more of the homing
molecules can comprise a methylated homing molecule. In some forms,
one or more of the methylated homing molecules can comprise a
methylated amino acid segment. In some forms, one or more of the
membrane perturbing molecules can be modified membrane perturbing
molecules. In some forms, one or more of the membrane perturbing
molecules comprise a methylated membrane perturbing molecule. In
some forms, one or more of the methylated membrane perturbing
molecules comprise a methylated amino acid segment. In some forms,
the amino acid sequence is N- or C-methylated in at least one
position.
[0075] In some forms, the composition can further comprise one or
more moieties. In some forms, the moieties can be independently
selected from the group consisting of an anti-angiogenic agent, a
pro-angiogenic agent, a cancer chemotherapeutic agent, a cytotoxic
agent, an anti-inflammatory agent, an anti-arthritic agent, a
polypeptide, a nucleic acid molecule, a small molecule, an image
contrast agent, a fluorophore, fluorescein, rhodamine, a
radionuclide, indium-111, technetium-99, carbon-11, and carbon-13.
In some forms, at least one of the moieties can be a therapeutic
agent. In some forms, the therapeutic agent can be iRGD, RGD,
Abraxane, paclitaxel, taxol, or a combination. In some forms, at
least one of the moieties can be a detectable agent. In some forms,
the detectable agent can be FAM.
[0076] In some forms, the composition can have a therapeutic
effect. In some forms, the composition can reduce tumor growth. In
some forms, the therapeutic effect can be a slowing in the increase
of or a reduction of tumor burden. In some forms, the therapeutic
effect can be a slowing of the increase of or reduction of tumor
size. In some forms, the subject can have one or more sites
targeted, wherein the composition can home to one or more of the
sites targeted. In some forms, the subject can have a tumor,
wherein the composition can have a therapeutic effect on the
tumor.
[0077] The disclosed components can be associated with each other
(or, in some forms, not associated with each other) in combinations
as disclosed herein. For example, homing molecules can be
covalently coupled or non-covalently associated with surface
molecules, homing molecules can be covalently coupled or
non-covalently associated with membrane perturbing molecules,
membrane perturbing molecules can be covalently coupled or
non-covalently associated with surface molecules, etc. Associated
components can also be referred to as being conjugated. Conjugation
can be direct or indirect. Direct conjugation of components refers
to covalently coupled or non-covalently associated components where
there is no other molecule intervening between the conjugated
components. Indirect conjugation refers to any chain of molecules
and covalent bonds or non-covalent associations linking the
components where the components are not directly conjugated (that
is, there is a least one separate molecule other than the
components intervening between the components).
[0078] Covalently coupled refers to association of components via
covalent bonds. A covalent association or coupling can be either
direct or indirect. A direct covalent association or coupling of
components refers to a covalent bond involving atoms that are each
respectively a part of the components. Thus, in a direct covalent
association or coupling, there is no other molecule intervening
between the associated/coupled components. An indirect covalent
association or coupling refers to any chain of molecules and
covalent bonds linking the components where the components are not
covalently coupled (that is, there is a least one separate molecule
other than the components intervening between the components via
covalent bonds).
[0079] As used herein, reference to components (such as a homing
molecule and a surface molecule) as being "not covalently coupled"
means that the components are not connected via covalent bonds (for
example, that the homing molecule and the surface molecule are not
connected via covalent bonds). That is, there is no continuous
chain of covalent bonds between, for example, the homing molecule
and the surface molecule.
[0080] Non-covalent association refers to association of components
via non-covalent bonds and interactions. A non-covalent association
can be either direct or indirect. A direct non-covalent association
refers to a non-covalent bond involving atoms that are each
respectively connected via a chain of covalent bonds to the
components. Thus, in a direct non-covalent association, there is no
other molecule intervening between the associated components. An
indirect non-covalent association refers to any chain of molecules
and bonds linking the components where the components are not
covalently coupled (that is, there is a least one separate molecule
other than the components intervening between the components via
non-covalent bonds).
[0081] Reference to components (such as a homing molecule and a
surface molecule) as not being "non-covalently associated" means
that there is no direct or indirect non-covalent association
between the components. That is, for example, no atom covalently
coupled to a homing molecule is involved in a non-covalent bond
with an atom covalently coupled to a surface molecule. Within this
meaning, a homing molecule and a surface molecule can be together
in a composition where they are indirectly associated via multiple
intervening non-covalent bonds while not being non-covalently
associated as that term is defined herein. For example, a homing
molecule and a surface molecule can be mixed together in a carrier
where they are not directly non-covalently associated. A homing
molecule and a surface molecule that are referred to as not
indirectly non-covalently associated cannot be mixed together in a
continuous composition. Reference to components (such as a homing
molecule and a surface molecule) as not being "directly
non-covalently associated" means that there is no direct
non-covalent association between the components (an indirect
non-covalent association may be present). Reference to components
(such as a homing molecule and a surface molecule) as not being
"indirectly non-covalently associated" means that there is no
direct or indirect non-covalent association between the
components.
[0082] It is understood that components can be non-covalently
associated via multiple chains and paths including both direct and
indirect non-covalent associations. For the purposes of these
definitions, the presence a single direct non-covalent association
makes the association a direct non-covalent association even if
there are also indirect non-covalent associations present.
Similarly, the presence of a covalent connection between components
means the components are covalently coupled even if there are also
non-covalent associations present. It is also understood that
covalently coupled components that happened to lack any
non-covalent association with each other are not considered to fall
under the definition of components that are not non-covalently
associated.
[0083] Association of the components of the disclosed compositions
can be aided or accomplished via molecules, conjugates and/or
compositions. Where such molecules, conjugates and/or compositions
are other than surface molecules, homing molecules, or cargo
molecules (such as membrane perturbing molecules, internalization
elements, tissue penetration elements, and moieties), they can be
referred to herein as linkers. Such linkers can be any molecule,
conjugate, composition, etc. that can be used to associate
components of the disclosed compositions. Generally, linkers can be
used to associate components other than surface molecules to
surface molecules. Useful linkers include materials that are
biocompatible, have low bioactivity, have low antigenicity, etc.
That is, such useful linker materials can serve the
linking/association function without adding unwanted bioreactivity
to the disclosed compositions. Many such materials are known and
used for similar linking and association functions. Polymer
materials are a particularly useful form of linker material. For
example, polyethylene glycols can be used.
[0084] Linkers are useful for achieving useful numbers and
densities of the components (such as homing molecules and membrane
perturbing molecules) on surface molecules. For example, linkers of
fibrous form are useful for increasing the number of components per
surface molecule or per a given area of the surface molecule.
Similarly, linkers having a branching form are useful for
increasing the number of components per surface molecule or per a
given area of the surface molecule. Linkers can also have a
branching fibrous form.
[0085] Sufficiency of the number and composition of homing
molecules in the composition can be determined by assessing homing
to the target and effectively delivery of the cargo molecules in a
non-human animal. The composition can comprise a sufficient number
and composition of homing molecules (modified or not) such that the
composition homes to the target and effectively delivers the cargo
molecules. In one example, sufficiency of the number and
composition of modified and/or unmodified homing molecules can be
determined by assessing cargo delivery and/or therapeutic effect on
the target. Sufficiency of the number and composition of membrane
perturbing molecules can be determined by assessing membrane
perturbing effect of the composition in a non-human animal. The
composition can comprise a sufficient number and composition of
membrane perturbing molecules (modified or not) such that the
composition has a membrane perturbing effect on the target. In one
example, sufficiency of the number and composition of modified
and/or unmodified membrane perturbing molecules can be determined
by assessing membrane disruption, apoptosis, and/or therapeutic
effect on the target.
[0086] The composition can comprise a sufficient density and
composition of homing molecules such that the composition homes to
the target and effectively delivers the cargo molecules.
Sufficiency of the density and composition of homing molecules can
be determined by assessing cargo delivery and/or therapeutic effect
on the target in a non-human animal. The composition can comprise a
sufficient density and composition of membrane perturbing molecules
such that the composition has a membrane perturbing effect on the
target. Sufficiency of the density and composition of membrane
perturbing molecules can be determined by assessing membrane
disruption, apoptosis, and/or therapeutic effect on the target in a
non-human animal.
[0087] The density of homing molecules and/or membrane perturbing
molecules on a surface molecule can be described in any suitable
manner For example, the density can be expressed as the number of
homing molecules and/or membrane perturbing molecules per, for
example, a given area, surface area, volume, unit, subunit, arm,
etc. of the surface molecule. The density can also be relative to,
for example, the area, surface area, volume, unit, subunit, arm,
etc. of the entire surface molecule or to the area, surface area,
volume, unit, subunit, arm, etc. of a portion of the surface
molecule. For example, a sufficient density of homing molecule
and/or membrane perturbing molecule can be present in a portion of
the surface molecule. The presence of this dense portion can cause
clotting and amplify the accumulation of the composition. Thus, a
composition having a sufficient density of homing molecules and/or
membrane perturbing molecules can have a threshold density (or
above) for the entire surface molecule or for just one or more
portions of the surface molecule. Unless otherwise stated,
densities refer to average density over the designated portion of
the surface molecule. For example, a density of 1 homing molecule
per square nM of the surface molecule refers to an average density
of the homing molecules over the entire surface molecule. As
another example, a density of 1 homing molecule per square nM of a
portion of the surface molecule refers to an average density of the
homing molecules over just that portion of the surface
molecule.
[0088] The density can be measured or calculated in any suitable
manner For example, the number or amount of homing molecules and/or
membrane perturbing molecules present on a surface molecule or
group of surface molecules can be measured by, for example,
detecting the level or intensity of signal produced by labeled
homing molecules and/or membrane perturbing molecules and
calculating the density based on the structural characteristics of
the surface molecule.
[0089] The density or threshold density of homing molecules and/or
membrane perturbing molecules can be, for example, at least 0.001,
0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 homing
molecules and/or membrane perturbing molecules per square nM of the
entire or a portion of the surface molecule. The composition can
also comprise any density in between those densities listed
above.
[0090] The density or threshold density of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules per square .mu.M of the entire or a
portion of the surface molecule. The composition can also comprise
any density in between those densities listed above.
[0091] The density or threshold density of homing molecules and/or
membrane perturbing molecules can be, for example, at least 0.001,
0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 homing
molecules and/or membrane perturbing molecules per cubic nM of the
entire or a portion of the surface molecule. The composition can
also comprise any density in between those densities listed
above.
[0092] The density or threshold density of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules per cubic .mu.M of the entire or a
portion of the surface molecule. The composition can also comprise
any density in between those densities listed above.
[0093] The number of homing molecules and/or membrane perturbing
molecules on a surface molecule can be described in any suitable
manner. For example, the number can be expressed as the number of
homing molecules and/or membrane perturbing molecules per, for
example, a given area, surface area, volume, unit, subunit, arm,
etc. of the surface molecule. The number can also be relative to,
for example, the area, surface area, volume, unit, subunit, arm,
etc. of the entire surface molecule or to the area, surface area,
volume, unit, subunit, arm, etc. of a portion of the surface
molecule. For example, a sufficient number of homing molecule
and/or membrane perturbing molecule can be present in a portion of
the surface molecule. The presence of this dense portion can cause
clotting and amplify the accumulation of the composition. Thus, a
composition having a sufficient number of homing molecules and/or
membrane perturbing molecules can have a threshold number (or
above) for the entire surface molecule or for just one or more
portions of the surface molecule.
[0094] The number can be measured or calculated in any suitable
manner For example, the number or amount of homing molecules and/or
membrane perturbing molecules present on a surface molecule or
group of surface molecules can be measured by, for example,
detecting the level or intensity of signal produced by labeled
homing molecules and/or membrane perturbing molecules and
calculating the number based on the structural characteristics of
the surface molecule.
[0095] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules on the surface molecule. The
composition can also comprise any number in between those numbers
listed above.
[0096] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 0.001,
0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 homing
molecules and/or membrane perturbing molecules per square nM of the
entire or a portion of the surface molecule. The composition can
also comprise any number in between those numbers listed above.
[0097] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules per square .mu.M of the entire or a
portion of the surface molecule. The composition can also comprise
any number in between those numbers listed above.
[0098] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 0.001,
0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 homing
molecules and/or membrane perturbing molecules per cubic nM of the
entire or a portion of the surface molecule. The composition can
also comprise any number in between those numbers listed above.
[0099] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules per cubic .mu.M of the entire or a
portion of the surface molecule. The composition can also comprise
any number in between those numbers listed above.
[0100] In some forms, the compositions not only home to tumors, but
also amplify their own homing. Homing molecules can be used that
are clot-binding compounds that recognize clotted plasma proteins
and selectively homes to tumors, where it binds to vessel walls and
tumor stroma. Surface molecules coupled with the clot-binding
compounds can accumulate in tumor vessels or at wound sites, where
they induce additional local clotting, thereby producing new
binding sites for more particles. The system mimics platelets,
which also circulate freely but accumulate at a diseased site and
amplify their own accumulation at that site. The clotting-based
amplification greatly enhances cargo delivery and tumor
imaging.
A. Homing Molecules
[0101] Homing molecules allow the disclosed compositions to be
targeted and to home to desired target sites. Homing molecules
generally bind preferentially to target molecules, cells, tissues,
etc., thus resulting in an accumulation of the homing molecules
(and other components to which they are associated) at target
sites.
[0102] The term "homing molecule" as used herein, means any
molecule that selectively homes in vivo to specified target sites,
such as cells or tissues, in preference to normal or other
non-target sites, cells, or tissues. Similarly, the term "homing
peptide" or "homing peptidomimetic" means a peptide that
selectively homes in vivo to specified target sites, such as cells
or tissues, in preference to normal or other non-target sites,
cells, or tissues. It is understood that a homing molecule that
selectively homes in vivo to, for example, tumors can home to all
tumors or can exhibit preferential homing to one or a subset of
tumor types.
[0103] By "selectively homes" it is meant that, in vivo, the homing
molecule binds preferentially to the target as compared to
non-target. For example, the homing molecule can bind
preferentially to certain molecules, proteins, cells, tissues, etc.
as compared to other molecules, proteins, cells, tissues, etc. For
example, the homing molecule can bind preferentially to tumor
vasculature or one or more tumors as compared to non-tumoral
tissue. Such a homing molecule can selectively home, for example,
to tumors. Selective homing to, for example, certain molecules,
proteins, cells, tissues, etc. generally is characterized by at
least a two-fold greater localization the molecules, proteins,
cells, tissues, etc. (or other target), as compared to other
certain molecules, proteins, cells, tissues, etc. A homing molecule
can be characterized by, for example, 5-fold, 10-fold, 20-fold or
more preferential localization to the target as compared to one or
more non-targets. For example, a homing molecule can be
characterized by, for example, 5-fold, 10-fold, 20-fold or more
preferential localization to tumor vasculature as compared to
vasculature of several or many tissue types of non-tumoral tissue,
or as compared to vasculature of most or all non-tumoral tissue. As
another example, a homing molecule can be characterized by, for
example, 5-fold, 10-fold, 20-fold or more preferential localization
to tumors as compared to several or many tissue types of
non-tumoral tissue, or as compared to-most or all non-tumoral
tissue. Thus, it is understood that, in some cases, a homing
molecule homes, in part, to one or more normal organs in addition
to homing to the target tissue. Selective homing can also be
referred to as targeting. The molecules, proteins, cells, tissues,
etc. that are targeted by homing molecules can be referred to as
targeted molecules, proteins, cells, tissues, etc.
[0104] In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative derivative thereof, the amino acid sequence CRKDKC
(SEQ ID NO:2) or a conservative derivative thereof, or a
combination. In some forms, one or more of the homing molecule can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative variant thereof. In some forms, one or more of the
homing molecules can comprise the amino acid sequence CGKRK (SEQ ID
NO:1).
[0105] The composition can comprise a sufficient number and
composition of homing molecules (modified or not) such that the
composition homes to the target and effectively delivers the cargo
molecules. In one example, sufficiency of the number and
composition of modified and/or unmodified homing molecules can be
determined by assessing cargo delivery and/or therapeutic effect on
the target.
[0106] Many homing molecules and homing peptides home to the
vasculature of the target tissue. However, for the sake of
convenience homing is referred to in some places herein as homing
to the tissue associated with the vasculature to which the homing
molecule or homing peptide may actually home. Thus, for example, a
homing molecule that homes to tumor vasculature can be referred to
herein as homing to tumor tissue or to tumor cells. By including or
associating a homing molecule or homing peptide with, for example,
a protein, peptide, amino acid sequence, cargo molecules, or CendR
element the protein, peptide, amino acid sequence, cargo molecules,
or CendR element can be targeted or can home to the target of the
homing molecule or homing peptide. In this way, the protein,
peptide, amino acid sequence, cargo molecules, or CendR element can
be said to home to the target of the homing molecule or homing
peptide. For convenience and unless otherwise indicated, reference
to homing of a protein, peptide, amino acid sequence, cargo
molecules, CendR element, etc. is intended to indicate that the
protein, peptide, amino acid sequence, cargo molecules, CendR
element, etc. includes or is associated with an appropriate homing
molecule or homing peptide.
[0107] The homing molecule can selectively home to a tumor. The
homing molecule can selectively home to tumor vasculature. The
homing molecule can selectively home to one or more particular
types of tumor. The homing molecule can selectively home to the
vasculature of one or more particular types of tumor. The homing
molecule can selectively home to one or more particular stages of a
tumor or cancer. The homing molecule can selectively home to the
vasculature of one or more particular stages of a tumor or cancer.
The homing molecule can selectively home to one or more particular
stages of one or more particular types of tumor. The homing
molecule can selectively home to the vasculature of one or more
different stages of one or more particular types of tumor.
[0108] The composition can selectively home to a tumor. The
composition can selectively home to tumor vasculature. The
composition can selectively home to one or more particular types of
tumor. The composition can selectively home to the vasculature of
one or more particular types of tumor. The composition can
selectively home to one or more particular stages of a tumor or
cancer. The composition can selectively home to the vasculature of
one or more particular stages of a tumor or cancer. The composition
can selectively home to one or more particular stages of one or
more particular types of tumor. The composition can selectively
home to the vasculature of one or more different stages of one or
more particular types of tumor.
[0109] The cargo molecule can selectively home to a tumor. The
cargo molecule can selectively home to tumor vasculature. The cargo
molecule can selectively home to one or more particular types of
tumor. The cargo molecule can selectively home to the vasculature
of one or more particular types of tumor. The cargo molecule can
selectively home to one or more particular stages of a tumor or
cancer. The cargo molecule can selectively home to the vasculature
of one or more particular stages of a tumor or cancer. The cargo
molecule can selectively home to one or more particular stages of
one or more particular types of tumor. The cargo molecule can
selectively home to the vasculature of one or more different stages
of one or more particular types of tumor.
[0110] The surface molecule can selectively home to a tumor. The
surface molecule can selectively home to tumor vasculature. The
surface molecule can selectively home to one or more particular
types of tumor. The surface molecule can selectively home to the
vasculature of one or more particular types of tumor. The surface
molecule can selectively home to one or more particular stages of a
tumor or cancer. The surface molecule can selectively home to the
vasculature of one or more particular stages of a tumor or cancer.
The surface molecule can selectively home to one or more particular
stages of one or more particular types of tumor. The surface
molecule can selectively home to the vasculature of one or more
different stages of one or more particular types of tumor.
[0111] The membrane perturbing molecule can selectively home to a
tumor. The membrane perturbing molecule can selectively home to
tumor vasculature. The membrane perturbing molecule can selectively
home to one or more particular types of tumor. The membrane
perturbing molecule can selectively home to the vasculature of one
or more particular types of tumor. The membrane perturbing molecule
can selectively home to one or more particular stages of a tumor or
cancer. The membrane perturbing molecule can selectively home to
the vasculature of one or more particular stages of a tumor or
cancer. The membrane perturbing molecule can selectively home to
one or more particular stages of one or more particular types of
tumor. The membrane perturbing molecule can selectively home to the
vasculature of one or more different stages of one or more
particular types of tumor.
[0112] The disclosed compositions, surface molecules, amino acid
sequences, cargo molecules, proteins or peptides can, for example,
home to brain cells, brain stem cells, brain tissue, and/or brain
vasculature, kidney cells, kidney stem cells, kidney tissue, and/or
kidney vasculature, skin cells, skin stem cells, skin tissue,
and/or skin vasculature, lung cells, lung tissue, and/or lung
vasculature, pancreatic cells, pancreatic tissue, and/or pancreatic
vasculature, intestinal cells, intestinal tissue, and/or intestinal
vasculature, adrenal gland cells, adrenal tissue, and/or adrenal
vasculature, retinal cells, retinal tissue, and/or retinal
vasculature, liver cells, liver tissue, and/or liver vasculature,
prostate cells, prostate tissue, and/or prostate vasculature,
endometriosis cells, endometriosis tissue, and/or endometriosis
vasculature, ovary cells, ovary tissue, and/or ovary vasculature,
tumor cells, tumors, tumor blood vessels, and/or tumor vasculature,
bone cells, bone tissue, and/or bone vasculature, bone marrow
cells, bone marrow tissue, and/or bone marrow vasculature,
cartilage cells, cartilage tissue, and/or cartilage vasculature,
stem cells, embryonic stem cells, pluripotent stem cells, induced
pluripotent stem cells, adult stem cells, hematopoietic stem cells,
neural stem cells, mesenchymal stem cells, mammary stem cells,
endothelial stem cells, olfactory adult stem cells, neural crest
stem cells, cancer stem cells, blood cells, erythrocytes,
platelets, leukocytes, granulocytes, neutrophils, eosinphils,
basophils, lymphoid cells, lymphocytes, monocytes, wound
vasculature, vasculature of injured tissue, vasculature of inflamed
tissue, atherosclerotic plaques, or a combination.
[0113] Examples of homing molecules and homing peptides are known.
Examples include: Brain homing peptides such as: CNSRLHLRC (SEQ ID
NO:8), CENWWGDVC (SEQ ID NO:9), WRCVLREGPAGGCAWFNRHRL (SEQ ID
NO:10), CLSSRLDAC (SEQ ID NO:11), CVLRGGRC (SEQ ID NO:12),
CNSRLQLRC (SEQ ID NO:13), CGVRLGC (SEQ ID NO:14), CKDWGRIC (SEQ ID
NO:15), CLDWGRIC (SEQ ID NO:16), CTRITESC (SEQ ID NO:17), CETLPAC
(SEQ ID NO:18), CRTGTLFC (SEQ ID NO:19), CGRSLDAC (SEQ ID NO:20),
CRHWFDVVC (SEQ ID NO:21), CANAQSHC (SEQ ID NO:22), CGNPSYRC (SEQ ID
NO:23), YPCGGEAVAGVSSVRTMCSE (SEQ ID NO:24), LNCDYQGTNPATSVSVPCTV
(SEQ ID NO:25); kidney homing peptides such as: CLPVASC (SEQ ID
NO:26), CGAREMC (SEQ ID NO:27), CKGRSSAC (SEQ ID NO:28), CWARAQGC
(SEQ ID NO:29), CLGRSSVC (SEQ ID NO:30), CTSPGGSC (SEQ ID NO:31),
CMGRWRLC (SEQ ID NO:32), CVGECGGC (SEQ ID NO:33), CVAWLNC (SEQ ID
NO:34), CRRFQDC (SEQ ID NO:35), CLMGVHC (SEQ ID NO:36), CKLLSGVC
(SEQ ID NO:37), CFVGHDLC (SEQ ID NO:38), CRCLNVC (SEQ ID NO:39),
CKLMGEC (SEQ ID NO:40); skin homing peptides such as: CARSKNKDC
(SEQ ID NO:41), CRKDKC (SEQ ID NO:42), CVALCREACGEGC (SEQ ID
NO:43), CSSGCSKNCLEMC (SEQ ID NO:44), CIGEVEVC (SEQ ID NO:45),
CKWSRLHSC (SEQ ID NO:46), CWRGDRKIC (SEQ ID NO:47), CERVVGSSC (SEQ
ID NO:48), CLAKENVVC (SEQ ID NO:49); lung homing peptides such as:
CGFECVRQCPERC (SEQ ID NO:50), CGFELETC (SEQ ID NO:51), CTLRDRNC
(SEQ ID NO:52), CIGEVEVC (SEQ ID NO:53), CTLRDRNC (SEQ ID NO:54),
CGKRYRNC (SEQ ID NO:55), CLRPYLNC (SEQ ID NO:56), CTVNEAYKTRMC (SEQ
ID NO:57), CRLRSYGTLSLC (SEQ ID NO:58), CRPWHNQAHTEC (SEQ ID
NO:59); pancreas homing peptides such as: SWCEPGWCR (SEQ ID NO:60),
CKAAKNK (SEQ ID NO:61), CKGAKAR (SEQ ID NO:62), VGVGEWSV (SEQ ID
NO:63); intestine homing peptides such as: YSGKWGW (SEQ ID NO:64);
uterus homing peptides such as: GLSGGRS (SEQ ID NO:65); adrenal
gland homing peptides such as: LMLPRAD (SEQ ID NO:66), LPRYLLS (SEQ
ID NO:67); retina homing peptides such as: CSCFRDVCC (SEQ ID
NO:68), CRDVVSVIC (SEQ ID NO:69); gut homing peptides such as:
YSGKWGK (SEQ ID NO:70), GISALVLS (SEQ ID NO:71), SRRQPLS (SEQ ID
NO:72), MSPQLAT (SEQ ID NO:73), MRRDEQR (SEQ ID NO:74), QVRRVPE
(SEQ ID NO:75), VRRGSPQ (SEQ ID NO:76), GGRGSWE (SEQ ID NO:77),
FRVRGSP (SEQ ID NO:78), RVRGPER (SEQ ID NO:79); liver homing
peptides such as: VKSVCRT (SEQ ID NO:80), WRQNMPL (SEQ ID NO:81),
SRRFVGG (SEQ ID NO:82), ALERRSL (SEQ ID NO:83), ARRGWTL (SEQ ID
NO:84); prostate homing peptides such as: SMSIARL (SEQ ID NO:85),
VSFLEYR (SEQ ID NO:86), RGRWLAL (SEQ ID NO:87); ovary homing
peptides such as: EVRSRLS (SEQ ID NO:88), VRARLMS (SEQ ID NO:89),
RVGLVAR (SEQ ID NO:90), RVRLVNL (SEQ ID NO:91); Clot binding/homing
peptide such as: CREKA (SEQ ID NO:92), CLOT1, and CLOT2; heart
homing peptides such as: CRPPR (SEQ ID NO:93), CGRKSKTVC (SEQ ID
NO:94), CARPAR (SEQ ID NO:95), CPKRPR (SEQ ID NO:96), CKRAVR (SEQ
ID NO:97), CRNSWKPNC (SEQ ID NO:98), RGSSS (SEQ ID NO:99),
CRSTRANPC (SEQ ID NO:100), CPKTRRVPC (SEQ ID NO:101), CSGMARTKC
(SEQ ID NO:102), GGGVFWQ (SEQ ID NO:103), HGRVRPH (SEQ ID NO:104),
VVLVTSS (SEQ ID NO:105), CLHRGNSC (SEQ ID NO:106), CRSWNKADNRSC
(SEQ ID NO:107), CGRKSKTVC (SEQ ID NO:108), CKRAVR (SEQ ID NO:109),
CRNSWKPNC (SEQ ID NO:110), CPKTRRVPC (SEQ ID NO:111), CSGMARTKC
(SEQ ID NO:112), CARPAR (SEQ ID NO:113), CPKRPR (SEQ ID NO:114);
tumor blood vessel homing peptide such as: CNGRC (SEQ ID NO:115)
and other peptides with the NGR motif (U.S. Pat. Nos. 6,177,542 and
6,576,239; U.S. Patent Application Publication No. 20090257951);
RGD peptides, and RGR peptides. Other homing peptides include
CSRPRRSEC (SEQ ID NO:116), CSRPRRSVC (SEQ ID NO:117), and CSRPRRSWC
(SEQ ID NO:118) (Hoffman et al., Cancer Cell, vol. 4 (2003)), F3
(KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK; SEQ ID NO:119), PQRRSARLSA (SEQ
ID NO:120), and PKRRSARLSA (SEQ ID NO:121) (U.S. Pat. No.
7,544,767).
[0114] Homing molecules can also be defined by their targets. For
example, numerous antigens and proteins are known that can be
useful for targeting. Any molecule that can bind, selectively bind,
home, selectively, target, selectively target, etc. such target
molecules can be used as a homing molecule. For example,
antibodies, nucleic acid aptamers, and compounds that can bind to
target molecules can be used as homing molecules. Examples of
useful target molecules for homing molecules include .alpha..nu.
integrins, .alpha..nu..beta.3 integrin, .alpha..nu..beta. 5
integrin, .alpha.5.beta.1 integrin, aminopeptidase N, tumor
endothelial markers (TEMs), endosialin, p32, gC1q receptor,
annexin-1, nucleolin, fibronectin ED-B, fibrin-fibronectin
complexes, interleukin-11 receptor .alpha., and protease-cleaved
collagen IV. These and other examples are described and referred to
in Ruoslahti et al., J. Cell Biology, 2010 (doi:
10.1083/jbc.200910104), which is hereby incorporated by reference
in its entirety and specifically for its description of and
references to target molecules.
[0115] The composition can comprise any number of homing molecules.
By way of example, the composition can comprise at least 1, 5, 10,
15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,
325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,
650, 675, 700, 625, 750, 775, 800, 825, 850, 875, 900, 925, 950,
975, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,
2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000,
6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000,
25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 75,000, or 100,000,
or more homing molecules. The composition can also comprise any
number in between those numbers listed above.
[0116] Homing molecules can be associated with and arranged in the
compositions in a variety of configurations. In some forms, homing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. In some forms, homing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of cargo molecules. In some forms, homing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of cargo molecules, wherein the cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. Combinations of these
combinations can also be used.
[0117] 1. Tumor-Homing Compounds
[0118] The disclosed homing molecules can be tumor-homing
compounds. Tumor-homing compounds are compounds that selectively
home to tumors and tumor-associated tissue. Many compounds that
target, bind to, and/or home to tumors are known, most of which can
be used as tumor-homing compounds in the disclosed compositions.
Tumor-homing compounds can each be independently selected from any
known tumor-homing compounds.
[0119] Tumor-homing compounds can comprise the amino acid sequence
CGKRK (SEQ ID NO:1) or a conservative derivative thereof, the amino
acid sequence CRKDKC (SEQ ID NO:2) or a conservative derivative
thereof, or a combination. Tumor-homing compounds can comprise the
amino acid sequence CGKRK (SEQ ID NO:1) or a conservative variant
thereof. In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1).
[0120] Useful peptides for tumor targeting include, for example,
the tumor-homing CendR peptide iRGD, LyP-1, a peptide that contains
a putative CendR element and has tumor-penetrating properties, and
RGR peptides. The LyP-1 peptide has a unique target within tumors;
it preferentially accumulates in the hypoxic/low nutrient areas of
tumors (Laakkonen et al., 2002; 2004; Karmali et al., 2009).
CRGRRST (SEQ ID NO:122; RGR; Joyce et al., 2003) is a peptide that
has been successfully used in targeting a cytokine antibody
combination into tumors (Hamzah et al., 2008). This peptide is
linear, which simplifies the synthesis. Like LyP-1, RGR is at least
to some extent tumor type-specific (Joyce et al., 2003), but the
tumor types recognized by the two peptides seem to be partially
different, which may be an advantage in testing combinations with
the pan-tumor iRGD.
[0121] Because tumors can include clot-related proteins, some
clot-binding and clot-homing compounds can also be tumor-homing
compounds. Such tumor-homing clot-binding compounds can be used as
tumor-homing compounds as described herein. Tumor-homing compounds
can each be independently selected from, for example, an amino acid
segment comprising the amino acid sequence REK, an amino acid
segment comprising the amino acid sequence CAR (such as CARSKNKDC
(SEQ ID NO:123)), an amino acid segment comprising the amino acid
sequence CRK (such as CRKDKC (SEQ ID NO:124)), a fibrin-binding
peptide, a peptide that binds clots and not fibrin (such as
CGLIIQKNEC (CLT1, SEQ ID NO:125) and CNAGESSKNC (CLT2, SEQ ID
NO:126)), a clot-binding antibody, and a clot-binding small organic
molecule. A plurality of the clot-binding compounds can each
independently comprise an amino acid segment comprising the amino
acid sequence REK. Such peptides are also described in U.S. Patent
Application Publication No. 2008/0305101, which is hereby
incorporated by reference for its description of such peptides.
Peptides comprising amino acid sequences CAR or CRK are also
described in U.S. Patent Application Publication No. 2009/0036349,
which is hereby incorporated by reference for its description of
such peptides.
[0122] LyP-1 are homing molecules that selectively home to tumor
lymphatic vasculature, for example, the lymphatic vasculature of
breast cancer tumors and osteosarcomas, in preference to normal
lymphatic vasculature. LyP-1 can selectively home, for example, to
the lymphatic vasculature of squamous carcinomas. The core LyP-1
peptide has an amino acid sequence CGNKRTRGC (SEQ ID NO:127). LyP-1
peptides are described in U.S. Patent Application Nos.
2004-0087499, 2007-0219134, and 2008-0014143, which are hereby
incorporated by reference in their entirety, an specifically for
their description of such peptides.
[0123] The clot-binding compound can also comprise a fibrin-binding
peptide (FBP). Examples of fibrin-binding peptides are known in the
art (Van Rooijen N, Sanders A (1994) J Immunol Methods 174: 83-93;
Moghimi S M, Hunter A C, Murray J C (2001) Pharmacol Rev 53:
283-318; U.S. Pat. No. 5,792,742, all herein incorporated by
reference in their entirety for their teaching concerning fibrin
binding peptides).
[0124] Clot-binding peptides can also bind to proteins other than
fibrin. Example include peptides that bind to fibronectin that has
become incorporated into a clot (Pilch et al., (2006) PNAS, 103:
2800-2804, hereby incorporated in its entirety for its teaching
concerning clot-binding peptides). Examples of clot-binding
peptides include, but are not limited to, CGLIIQKNEC (CLT1, SEQ ID
NO:125) and CNAGESSKNC (CLT2, SEQ ID NO:126). The amino acid
segments can also be independently selected from amino acid
segments comprising the amino acid sequence CLT1 or CLT2 or a
conservative variant thereof, amino acid segments comprising the
amino acid sequence CLT1 or CLT2, or amino acid segments consisting
of the amino acid sequence CLT1 or CLT2. The amino acid segments
can each independently comprise the amino acid sequence CLT1 or
CLT2 or a conservative variant thereof. The amino acid segments can
also each independently comprise the amino acid sequence CLT1 or
CLT2. The amino acid segment can also consist of the amino acid
sequence CLT1 or CLT2.
[0125] The amino acid segments can also each independently comprise
the amino acid sequence CARSKNKDC (SEQ ID NO:128), and the amino
acid sequence CRK (such as CRKDKC (SEQ ID NO:129). Peptides
comprising amino acid sequences CAR or CRK are also described in
U.S. Patent Application Publication No. 2009/0036349, which is
hereby incorporated by reference for its description of such
peptides.
[0126] The composition can comprise any number of tumor-homing
compounds. By way of example, the composition can comprise at least
1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250,
275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,
600, 625, 650, 675, 700, 625, 750, 775, 800, 825, 850, 875, 900,
925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000,
15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000,
75,000, or 100,000, or more tumor-homing compounds. The composition
can also comprise any number in between those numbers listed
above.
[0127] Table 1 shows examples of tumor-homing CendR peptides.
TABLE-US-00001 TABLE 1 Examples of Tumor-Homing Peptides with CendR
Elements Sequence Reference CRKDKC (SEQ ID NO: 42) Jarvinen et al.,
Am. J. Pathol. 171(2): 702-711 (2007) CGNKRTRGC (SEQ ID Laakkonen
et al., Nature Medicine 8: 751-755 NO: 127) (2002)
AKVKDEPQRRSARLSAK Christian et al., JCB, 163(4): 871-878 (2003);
PAPPKPEPKPKKAPAKK U.S. Pat. No. 7,544,767 (SEQ ID NO: 137)
CSRPRRSEC (SEQ ID Hoffman et al., Cancer Cell, vol. 4 (2003) NO:
116) CSRPRRSVC (SEQ ID NO: 117) CSRPRRSWC (SEQ ID NO: 118)
CNRRTKAGC (SEQ ID Zhang et al., Cancer Res. 66(11): 5696-5706 NO:
132) (2006) CRGRRST (SEQ ID Joyce et al., Cancer Cell, 4(5):
393-403 (2003) NO: 122) CRSRKG (SEQ ID NO: 133) CKAAKNK (SEQ ID NO:
61) CKGAKAR (SEQ ID NO: 62) PQRRSARLSA (SEQ ID Porkka et al., Proc.
Natl. Acad. Sci. USA NO: 120) 99(11): 7444-7449 (2002); U.S. Pat.
No. 7,544,767 PKRRSARLSA (SEQ ID U.S. Pat. No. 7,544,767 NO: 121)
CRGDKGPDC (SEQ ID iRGD, Sugahara et al., Cancer Cell (2009); NO:
134) Sugahara et al. Science (2010); U.S. patent application No.
12/355,672, filed Jan. 19, 2009
[0128] Tumor-homing compounds can also be modified. Any of the
modifications described herein for homing molecules can be used
with the disclosed tumor-homing compounds.
[0129] 2. Modified Homing Molecules
[0130] The disclosed homing molecules can include modified forms of
homing molecules. The homing molecules can have any useful
modification. For example, some modifications can stabilize the
homing molecule. For example, the disclosed homing molecules
include methylated homing molecules. Methylated homing molecules
are particularly useful when the homing molecule includes a
protein, peptide or amino acid segment. For example, a homing
molecule can be a modified homing molecule, where, for example, the
modified homing molecule includes a modified amino acid segment or
amino acid sequence. For example, a modified homing molecule can be
a methylated homing molecule, where, for example, the methylated
homing molecule includes a methylated amino acid segment or amino
acid sequence. Other modifications can be used, either alone or in
combination. Where the homing molecule is, or includes, a protein,
peptide, amino acid segment and/or amino acid sequences, the
modification can be to the protein, peptide, amino acid segment,
amino acid sequences and/or any amino acids in the protein,
peptide, amino acid segment and/or amino acid sequences Amino acid
and peptide modifications are known to those of skill in the art,
some of which are described below and elsewhere herein. Methylation
is a particularly useful modification for the disclosed homing
molecules. Using modified forms of homing molecules can increase
the effectiveness of the homing and targeting, which can increase
the effect on the target.
[0131] A plurality of modified and/or unmodified homing molecules
can each be independently selected from, for example, an amino acid
segment comprising a modified or unmodified form of the amino acid
sequence of a homing peptide, an amino acid segment comprising a
modified or unmodified form of the amino acid sequence CGKRK (SEQ
ID NO:1), and an amino acid segment comprising a modified or
unmodified form of the amino acid sequence CRKDKC (SEQ ID NO:2). A
plurality of the homing molecules can each independently comprise
an amino acid segment comprising a modified or unmodified form of
the amino acid sequence of a homing peptide.
[0132] The composition can comprise any number of modified and/or
unmodified homing molecules. By way of example, the composition can
comprise at least 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
525, 550, 575, 600, 625, 650, 675, 700, 625, 750, 775, 800, 825,
850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500,
1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750, 3000, 3500, 4000,
4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,
10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000,
50,000, 75,000, or 100,000, or more modified and/or unmodified
homing molecules. The composition can also comprise any number in
between those numbers listed above.
[0133] As used herein, a "methylated derivative" of a protein,
peptide, amino acid segment, amino acid sequence, etc. refers to a
form of the protein, peptide, amino acid segment, amino acid
sequence, etc. that is methylated. Unless the context indicates
otherwise, reference to a methylated derivative of a protein,
peptide, amino acid segment, amino acid sequence, etc. does no
include any modification to the base protein, peptide, amino acid
segment, amino acid sequence, etc. other than methylation.
Methylated derivatives can also have other modifications, but such
modifications generally will be noted. For example, conservative
variants of an amino acid sequence would include conservative amino
acid substitutions of the base amino acid sequence. Thus, reference
to, for example, a "methylated derivative" of a specific amino acid
sequence "and conservative variants thereof" would include
methylated forms of the specific amino acid sequence and methylated
forms of the conservative variants of the specific amino acid
sequence, but not any other modifications of derivations. As
another example, reference to a methylated derivative of an amino
acid segment that includes amino acid substitutions would include
methylated forms of the amino acid sequence of the amino acid
segment and methylated forms of the amino acid sequence of the
amino acid segment include amino acid substitutions.
B. Cargo Molecules
[0134] The disclosed compositions include one or more cargo
molecules. Generally, the disclosed compositions can include a
plurality of cargo molecules. The disclosed compositions can
include a single type of cargo molecule or a plurality of different
types of cargo molecules. Thus, for example, the disclosed
compositions can include a plurality of different types of cargo
molecules where a plurality of one or more of the different types
of cargo molecules can be present.
[0135] Cargo molecules can be any compound, molecule, conjugate,
composition, etc. that is desired to be delivered using the
disclosed compositions. For example, the cargo molecules can be
therapeutic agents, detectable agents, or a combination. For
example, the cargo molecules can be membrane perturbing molecules,
pro-apoptotic molecules, pore-generating molecules, antimicrobial
molecules, mitochondria-affecting molecules, mitochondria-targeted
molecules, or a combination. Examples of some useful cargo
molecules are described below and elsewhere herein.
[0136] Cargo molecules can be associated with and arranged in the
compositions in a variety of configurations. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of homing molecules. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of homing molecules, wherein the homing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. Combinations of these
combinations can also be used.
[0137] 1. Membrane Perturbing Molecules
[0138] Useful forms of cargo molecules include membrane perturbing
molecules. Membrane perturbing molecules include molecules that can
disrupt membranes, that can form pores in membranes, that can make
membranes leaky, that can be targeted to or affect intracellular
membranes or organelles, such mitochondria or lysosomes. Some forms
of membrane perturbing molecules can be pro-apoptotic while others
can be non-apoptotic. Some forms of membrane perturbing molecules
can be pro-apoptotic for only some types of cells.
[0139] In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1). In some
forms, one or more of the membrane perturbing molecules can
comprise the amino acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID
NO:3) or a conservative variant thereof, (KLAKLAK).sub.2 (SEQ ID
NO:3) or a conservative variant thereof, (KLAKKLA).sub.2 (SEQ ID
NO:5) or a conservative variant thereof, (KAAKKAA).sub.2 (SEQ ID
NO:6) or a conservative variant thereof, (KLGKKLG).sub.3 (SEQ ID
NO:7) or a conservative variant thereof, or a combination. In some
forms, one or more of the membrane perturbing molecules can
comprise the amino acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID
NO:3), (KLAKLAK).sub.2 (SEQ ID NO:3), (KLAKKLA).sub.2 (SEQ ID
NO:5), (KAAKKAA).sub.2 (SEQ ID NO:6), (KLGKKLG).sub.3 (SEQ ID
NO:7), or a combination. In some forms, one or more of the membrane
perturbing molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative variant
thereof. In some forms, one or more of the membrane perturbing
molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3). Membrane perturbing peptides
of this type are described in Ellerby, Nature Medicine 5, 1032-1038
(1999), which is hereby incorporated by reference for its
description of such peptides.
[0140] A plurality of modified and/or unmodified membrane
perturbing molecules can each be independently selected from, for
example, an amino acid segment comprising a modified or unmodified
form of the amino acid sequence of a homing peptide, an amino acid
segment comprising a modified or unmodified form of the amino acid
sequence .sub.D(KLAKLAK).sub.2 (SEQ ID NO:3), (KLAKLAK).sub.2 (SEQ
ID NO:3), (KLAKKLA).sub.2 (SEQ ID NO:5), (KAAKKAA).sub.2 (SEQ ID
NO:6), (KLGKKLG).sub.3 (SEQ ID NO:7), or a combination. A plurality
of the membrane perturbing molecules can each independently
comprise an amino acid segment comprising a modified or unmodified
form of the amino acid sequence of a homing peptide.
[0141] The composition can comprise a sufficient number and
composition of membrane perturbing molecules (modified or not) such
that the composition has a membrane perturbing effect on the
target. In one example, sufficiency of the number and composition
of modified and/or unmodified membrane perturbing molecules can be
determined by assessing membrane disruption, apoptosis, and/or
therapeutic effect on the target.
[0142] The composition can comprise any number of modified and/or
unmodified membrane perturbing molecules. By way of example, the
composition can comprise at least 1, 5, 10, 15, 20, 25, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,
425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 625,
750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200,
1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750,
3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,
8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000,
40,000, 45,000, 50,000, 75,000, or 100,000, or more modified and/or
unmodified membrane perturbing molecules. The composition can also
comprise any number in between those numbers listed above.
[0143] Membrane perturbing molecules can be associated with and
arranged in the compositions in a variety of configurations. In
some forms, membrane perturbing molecules can be associated with,
conjugated to, and/or covalently coupled to a plurality of surface
molecules. In some forms, membrane perturbing molecules can be
associated with, conjugated to, and/or covalently coupled to a
plurality of homing molecules. In some forms, membrane perturbing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of homing molecules, wherein the homing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. Combinations of these
combinations can also be used. [0144] i. Modified Membrane
Perturbing Molecules
[0145] The disclosed membrane perturbing molecules can include
modified forms of membrane perturbing molecules. The membrane
perturbing molecules can have any useful modification. For example,
some modifications can stabilize the membrane perturbing molecule.
For example, the disclosed membrane perturbing molecules include
methylated membrane perturbing molecules. Methylated membrane
perturbing molecules are particularly useful when the membrane
perturbing molecule includes a protein, peptide or amino acid
segment. For example, a membrane perturbing molecule can be a
modified membrane perturbing molecule, where, for example, the
modified membrane perturbing molecule includes a modified amino
acid segment or amino acid sequence. For example, a modified
membrane perturbing molecule can be a methylated membrane
perturbing molecule, where, for example, the methylated membrane
perturbing molecule includes a methylated amino acid segment or
amino acid sequence. Other modifications can be used, either alone
or in combination. Where the membrane perturbing molecule is, or
includes, a protein, peptide, amino acid segment and/or amino acid
sequences, the modification can be to the protein, peptide, amino
acid segment, amino acid sequences and/or any amino acids in the
protein, peptide, amino acid segment and/or amino acid sequences
Amino acid and peptide modifications are known to those of skill in
the art, some of which are described below and elsewhere herein.
Methylation is a particularly useful modification for the disclosed
membrane perturbing molecules. Using modified forms of membrane
perturbing molecules can increase their effectiveness.
[0146] 2. Moieties
[0147] The disclosed compositions can further comprise one or more
moieties. The cargo molecules of the disclosed compositions can
include one or more moieties. For example, the moieties can be
independently selected from the group consisting of an
anti-angiogenic agent, a pro-angiogenic agent, a cancer
chemotherapeutic agent, a cytotoxic agent, an anti-inflammatory
agent, an anti-arthritic agent, a polypeptide, a nucleic acid
molecule, a small molecule, an image contrast agent, a fluorophore,
fluorescein, rhodamine, a radionuclide, indium-111, technetium-99,
carbon-11, and carbon-13. In some forms, at least one of the
moieties can be a therapeutic agent. Examples of therapeutic agents
are paclitaxel and taxol. In some forms, at least one of the
moieties can be a detectable agent.
[0148] As used herein, the term "moiety" is used broadly to mean a
physical, chemical, or biological material that generally imparts a
biologically useful function to a linked or conjugated molecule. As
disclosed herein, the properties of the moiety can also be found in
a surface molecule, or both the surface molecule and the moiety can
share one of the traits disclosed herein. For example, the surface
molecule can comprise a detectable agent, while the moiety can
comprise a therapeutic agent. This also applies for the homing
molecules, which can also comprise one or more of the properties of
moieties as disclosed herein. The description of therapeutic and
detectable agents herein is intended to apply to any of the
disclosed cargo molecules, membrane perturbing molecules, moieties,
surface molecules, or homing molecules. Thus, for example, moieties
can be conjugated to, coupled to, or can be part of, for example,
the disclosed surface molecules, homing molecules, membrane
perturbing molecules, or compositions comprising, or conjugates of,
surface molecules, homing molecules, and membrane perturbing
molecules.
[0149] A moiety can be any natural or non-natural material
including, without limitation, a biological material, such as a
cell, phage or other virus; an organic chemical such as a small
molecule; a radionuclide; a nucleic acid molecule or
oligonucleotide; a polypeptide; or a peptide. Useful moieties
include, but are not limited to, therapeutic agents such as cancer
chemotherapeutic agents, cytotoxic agents, pro-apoptotic agents,
and anti-angiogenic agents; detectable labels and imaging agents;
and tags or other insoluble supports. Useful moieties further
include, without limitation, phage and other viruses, cells,
liposomes, polymeric matrices, non-polymeric matrices or particles
such as gold particles, microdevices and nanodevices, and
nano-scale semiconductor materials. These and other moieties known
in the art can be components of a composition.
[0150] In some forms, the moiety can be an RGD peptide, such as
iRGD. iRGD peptides and their use are described in U.S. Patent
Application Publication 2009-0246133, which is hereby incorporated
by reference in its entirety, and specifically for its description
of the form, structure, and use of iRGD. [0151] i. Therapeutic
Agents
[0152] The moiety can be a therapeutic agent. As used herein, the
term "therapeutic agent" means a molecule which can have one or
more biological activities in a normal or pathologic tissue. A
variety of therapeutic agents can be used as a moiety. The
therapeutic agent can comprise a compound or composition for
treating cancer. The therapeutic agent can comprise a compound or
composition to induce programmed cell death or apoptosis. Membrane
perturbing molecules are a form of therapeutic agent.
[0153] In some embodiments, the therapeutic agent can be a cancer
chemotherapeutic agent. As used herein, a "cancer chemotherapeutic
agent" is a chemical agent that inhibits the proliferation, growth,
life-span or metastatic activity of cancer cells. Such a cancer
chemotherapeutic agent can be, without limitation, a taxane such as
docetaxel; an anthracyclin such as doxorubicin; an alkylating
agent; a vinca alkaloid; an anti-metabolite; a platinum agent such
as cisplatin or carboplatin; a steroid such as methotrexate; an
antibiotic such as adriamycin; a isofamide; or a selective estrogen
receptor modulator; an antibody such as trastuzumab.
[0154] Taxanes are chemotherapeutic agents useful with the
compositions disclosed herein. Useful taxanes include, without
limitation, docetaxel (Taxotere; Aventis Pharmaceuticals, Inc.;
Parsippany, N.J.) and paclitaxel (Taxol; Bristol-Myers Squibb;
Princeton, N.J.). See, for example, Chan et al., J. Clin. Oncol.
17:2341-2354 (1999), and Paridaens et al., J. Clin. Oncol. 18:724
(2000).
[0155] A cancer chemotherapeutic agent useful with the compositions
disclosed herein also can be an anthracyclin such as doxorubicin,
idarubicin or daunorubicin. Doxorubicin is a commonly used cancer
chemotherapeutic agent and can be useful, for example, for treating
breast cancer (Stewart and Ratain, In: "Cancer: Principles and
practice of oncology" 5th ed., chap. 19 (eds. DeVita, Jr., et al.;
J. P. Lippincott 1997); Harris et al., In "Cancer: Principles and
practice of oncology," supra, 1997). In addition, doxorubicin has
anti-angiogenic activity (Folkman, Nature Biotechnology 15:510
(1997); Steiner, In "Angiogenesis: Key principles-Science,
technology and medicine," pp. 449-454 (eds. Steiner et al.;
Birkhauser Verlag, 1992)), which can contribute to its
effectiveness in treating cancer.
[0156] An alkylating agent such as melphalan or chlorambucil also
can be a useful cancer chemotherapeutic agent. Similarly, a vinca
alkaloid such as vindesine, vinblastine or vinorelbine; or an
antimetabolite such as 5-fluorouracil, 5-fluorouridine or a
derivative thereof can be a useful cancer chemotherapeutic
agent.
[0157] A platinum agent also can be a useful cancer
chemotherapeutic agent. Such a platinum agent can be, for example,
cisplatin or carboplatin as described, for example, in Crown,
Seminars in Oncol. 28:28-37 (2001). Other useful cancer
chemotherapeutic agents include, without limitation, methotrexate,
mitomycin-C, adriamycin, ifosfamide and ansamycins.
[0158] A cancer chemotherapeutic agent useful for treatment of
breast cancer and other hormonally-dependent cancers also can be an
agent that antagonizes the effect of estrogen, such as a selective
estrogen receptor modulator or an anti-estrogen. The selective
estrogen receptor modulator, tamoxifen, is a cancer
chemotherapeutic agent that can be used in a composition for
treatment of breast cancer (Fisher et al., J. Natl. Cancer Instit.
90:1371-1388 (1998)).
[0159] The therapeutic agent can be an antibody such as a humanized
monoclonal antibody. As an example, the anti-epidermal growth
factor receptor 2 (HER2) antibody, trastuzumab (Herceptin;
Genentech, South San Francisco, Calif.) can be a therapeutic agent
useful for treating HER2/neu overexpressing breast cancers (White
et al., Annu. Rev. Med. 52:125-141 (2001)).
[0160] Useful therapeutic agents also can be a cytotoxic agent,
which, as used herein, can be any molecule that directly or
indirectly promotes cell death. Useful cytotoxic agents include,
without limitation, small molecules, polypeptides, peptides,
peptidomimetics, nucleic acid-molecules, cells and viruses. As
non-limiting examples, useful cytotoxic agents include cytotoxic
small molecules such as doxorubicin, docetaxel or trastuzumab;
antimicrobial peptides such as those described further below;
pro-apoptotic polypeptides such as caspases and toxins, for
example, caspase-8; diphtheria toxin A chain, Pseudomonas exotoxin
A, cholera toxin, ligand fusion toxins such as DAB389EGF, ricinus
communis toxin (ricin); and cytotoxic cells such as cytotoxic T
cells. See, for example, Martin et al., Cancer Res. 60:3218-3224
(2000); Kreitman and Pastan, Blood 90:252-259 (1997); Allam et al.,
Cancer Res. 57:2615-2618 (1997); and Osborne and Coronado-Heinsohn,
Cancer J. Sci. Am. 2:175 (1996). One skilled in the art understands
that these and additional cytotoxic agents described herein or
known in the art can be useful in the disclosed compositions and
methods.
[0161] In one embodiment, a therapeutic agent can be a therapeutic
polypeptide. As used herein, a therapeutic polypeptide can be any
polypeptide with a biologically useful function. Useful therapeutic
polypeptides encompass, without limitation, cytokines, antibodies,
cytotoxic polypeptides; pro-apoptotic polypeptides; and
anti-angiogenic polypeptides. As non-limiting examples, useful
therapeutic polypeptides can be a cytokine such as tumor necrosis
factor-.alpha. (TNF-.alpha.), tumor necrosis factor-.beta.
(TNF-.beta.), granulocyte macrophage colony stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF),
interferon-.alpha. (IFN-.alpha.); interferon-.gamma. (IFN-.gamma.),
interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3),
interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7),
interleukin-10 (IL-10), interleukin-12 (IL-12), lymphotactin (LTN)
or dendritic cell chemokine 1 (DC-CK1); an anti-HER2 antibody or
fragment thereof; a cytotoxic polypeptide including a toxin or
caspase, for example, diphtheria toxin A chain, Pseudomonas
exotoxin A, cholera toxin, a ligand fusion toxin such as DAB389EGF
or ricin; or an anti-angiogenic polypeptide such as angiostatin,
endostatin, thrombospondin, platelet factor 4; anastellin; or one
of those described further herein or known in the art (see below).
It is understood that these and other polypeptides with biological
activity can be a "therapeutic polypeptide."
[0162] A therapeutic agent can also be an anti-angiogenic agent. As
used herein, the term "anti-angiogenic agent" means a molecule that
reduces or prevents angiogenesis, which is the growth and
development of blood vessels. A variety of anti-angiogenic agents
can be prepared by routine methods. Such anti-angiogenic agents
include, without limitation, small molecules; proteins such as
dominant negative forms of angiogenic factors, transcription
factors and antibodies; peptides; and nucleic acid molecules
including ribozymes, antisense oligonucleotides, and nucleic acid
molecules encoding, for example, dominant negative forms of
angiogenic factors and receptors, transcription factors, and
antibodies and antigen-binding fragments thereof. See, for example,
Hagedorn and Bikfalvi, Crit. Rev. Oncol. Hematol. 34:89-110 (2000),
and Kirsch et al., J. Neurooncol. 50:149-163 (2000).
[0163] Vascular endothelial growth factor (VEGF) has been shown to
be important for angiogenesis in many types of cancer, including
breast cancer angiogenesis in vivo (Borgstrom et al., Anticancer
Res. 19:4213-4214 (1999)). The biological effects of VEGF include
stimulation of endothelial cell proliferation, survival, migration
and tube formation, and regulation of vascular permeability. An
anti-angiogenic agent can be, for example, an inhibitor or
neutralizing antibody that reduces the expression or signaling of
VEGF or another angiogenic factor, for example, an anti-VEGF
neutralizing monoclonal antibody (Borgstrom et al., supra, 1999).
An anti-angiogenic agent also can inhibit another angiogenic factor
such as a member of the fibroblast growth factor family such as
FGF-1 (acidic), FGF-2 (basic), FGF-4 or FGF-5 (Slavin et al., Cell
Biol. Int. 19:431-444 (1995); Folkman and Shing, J. Biol. Chem.
267:10931-10934 (1992)) or an angiogenic factor such as
angiopoietin-1, a factor that signals through the endothelial
cell-specific Tie2 receptor tyrosine kinase (Davis et al., Cell
87:1161-1169 (1996); and Suri et al., Cell 87:1171-1180 (1996)), or
the receptor of one of these angiogenic factors. It is understood
that a variety of mechanisms can act to inhibit activity of an
angiogenic factor including, without limitation, direct inhibition
of receptor binding, indirect inhibition by reducing secretion of
the angiogenic factor into the extracellular space, or inhibition
of expression, function or signaling of the angiogenic factor.
[0164] A variety of other molecules also can function as
anti-angiogenic agents including, without limitation, angiostatin;
a kringle peptide of angiostatin; endostatin; anastellin,
heparin-binding fragments of fibronectin; modified forms of
antithrombin; collagenase inhibitors; basement membrane turnover
inhibitors; angiostatic steroids; platelet factor 4 and fragments
and peptides thereof; thrombospondin and fragments and peptides
thereof; and doxorubicin (O'Reilly et al., Cell 79:315-328 (1994));
O'Reilly et al., Cell 88:277-285 (1997); Homandberg et al., Am. J.
Path. 120:327-332 (1985); Homandberg et-al., Biochim Biophys. Acta
874:61-71 (1986); and O'Reilly et al., Science 285:1926-1928
(1999)). Commercially available anti-angiogenic agents include, for
example, angiostatin, endostatin, metastatin and 2ME2 (EntreMed;
Rockville, Md.); anti-VEGF antibodies such as Avastin (Genentech;
South San Francisco, Calif.); and VEGFR-2 inhibitors such as
SU5416, a small molecule inhibitor of VEGFR-2 (SUGEN; South San
Francisco, Calif.) and SU6668 (SUGEN), a small molecule inhibitor
of VEGFR-2, platelet derived growth factor and fibroblast growth
factor I receptor. It is understood that these and other
anti-angiogenic agents can be prepared by routine methods and are
encompassed by the term "anti-angiogenic agent" as used herein.
[0165] The compositions disclosed herein can also be used at a site
of inflammation or injury. Moieties useful for this purpose can
include therapeutic agents belonging to several basic groups
including anti-inflammatory agents which prevent inflammation,
restenosis preventing drugs which prevent tissue growth,
anti-thrombogenic drugs which inhibit or control formation of
thrombus or thrombolytics, and bioactive agents which regulate
tissue growth and enhance healing of the tissue. Examples of useful
therapeutic agents include but are not limited to steroids,
fibronectin, anti-clotting drugs, anti-platelet function drugs,
drugs which prevent smooth muscle cell growth on inner surface wall
of vessel, heparin, heparin fragments, aspirin, coumadin, tissue
plasminogen activator (TPA), urokinase, hirudin, streptokinase,
antiproliferatives (methotrexate, cisplatin, fluorouracil,
Adriamycin), antioxidants (ascorbic acid, beta carotene, vitamin
E), antimetabolites, thromboxane inhibitors, non-steroidal and
steroidal anti-inflammatory drugs, beta and calcium channel
blockers, genetic materials including DNA and RNA fragments,
complete expression genes, antibodies, lymphokines, growth factors,
prostaglandins, leukotrienes, laminin, elastin, collagen, and
integrins.
[0166] Useful therapeutic agents also can be antimicrobial
peptides. Thus, for example, also disclosed are moieties comprising
an antimicrobial peptide, where the composition is selectively
internalized and exhibits a high toxicity to the targeted area.
Useful antimicrobial peptides can have low mammalian cell toxicity
when not incorporated into the composition. As used herein, the
term "antimicrobial peptide" means a naturally occurring or
synthetic peptide having antimicrobial activity, which is the
ability to kill or slow the growth of one or more microbes. An
antimicrobial peptide can, for example, kill or slow the growth of
one or more strains of bacteria including a Gram-positive or
Gram-negative bacteria, or a fungi or protozoa. Thus, an
antimicrobial peptide can have, for example, bacteriostatic or
bacteriocidal activity against, for example, one or more strains of
Escherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus.
While not wishing to be bound by the following, an antimicrobial
peptide can have biological activity due to the ability to form ion
channels through membrane bilayers as a consequence of
self-aggregation.
[0167] An antimicrobial peptide is typically highly basic and can
have a linear or cyclic structure. As discussed further below, an
antimicrobial peptide can have an amphipathic .alpha.-helical
structure (see U.S. Pat. No. 5,789,542; Javadpour et al., J. Med.
Chem. 39:3107-3113 (1996); and Blondelle and Houghten, Biochem. 31:
12688-12694 (1992)). An antimicrobial peptide also can be, for
example, a .beta.-strand/sheet-forming peptide as described in
Mancheno et al., J. Peptide Res. 51:142-148 (1998).
[0168] An antimicrobial peptide can be a naturally occurring or
synthetic peptide. Naturally occurring antimicrobial peptides have
been isolated from biological sources such as bacteria, insects,
amphibians, and mammals and are thought to represent inducible
defense proteins that can protect the host organism from bacterial
infection. Naturally occurring antimicrobial peptides include the
gramicidins, magainins, mellitins, defensins and cecropins (see,
for example, Maloy and Kari, Biopolymers 37:105-122 (1995);
Alvarez-Bravo et al., Biochem. J. 302:535-538 (1994); Bessalle et
al., FEBS 274:-151-155 (1990.); and Blondelle and Houghten in
Bristol (Ed.), Annual Reports in Medicinal Chemistry pages 159-168
Academic Press, San Diego). An antimicrobial peptide also can be an
analog of a natural peptide, especially one that retains or
enhances amphipathicity (see below).
[0169] An antimicrobial peptide incorporated into the composition
disclosed herein can have low mammalian cell toxicity when linked
to the composition. Mammalian cell toxicity readily can be assessed
using routine assays. As an example, mammalian cell toxicity can be
assayed by lysis of human erythrocytes in vitro as described in
Javadpour et al., supra, 1996. An antimicrobial peptide having low
mammalian cell toxicity is not lytic to human erythrocytes or
requires concentrations of greater than 100 .mu.M for lytic
activity, preferably concentrations greater than 200, 300, 500 or
1000 .mu.M.
[0170] In one embodiment, disclosed are compositions in which the
antimicrobial peptide portion promotes disruption of mitochondrial
membranes when internalized by eukaryotic cells. In particular,
such an antimicrobial peptide preferentially disrupts mitochondrial
membranes as compared to eukaryotic membranes. Mitochondrial
membranes, like bacterial membranes but in contrast to eukaryotic
plasma membranes, have a high content of negatively charged
phospholipids. An antimicrobial peptide can be assayed for activity
in disrupting mitochondrial membranes using, for example, an assay
for mitochondrial swelling or another assay well known in the
art.
[0171] An antimicrobial peptide that induces significant
mitochondrial swelling at, for example, 50 .mu.M, 40 .mu.M, 30
.mu.M, 20 .mu.M, 10 .mu.M, or less, is considered a peptide that
promotes disruption of mitochondrial membranes.
[0172] Antimicrobial peptides generally have random coil
conformations in dilute aqueous solutions, yet high levels of
helicity can be induced by helix-promoting solvents and amphipathic
media such as micelles, synthetic bilayers or cell membranes.
.alpha.-Helical structures are well known in the art, with an ideal
.alpha.-helix characterized by having 3.6 residues per turn and a
translation of 1.5 .ANG. per residue (5.4 .ANG. per turn; see
Creighton, Proteins: Structures and Molecular Properties W. H
Freeman, New York (1984)). In an amphipathic .alpha.-helical
structure, polar and non-polar amino acid residues are aligned into
an amphipathic helix, which is a .alpha.-helix in which the
hydrophobic amino acid residues are predominantly on one face, with
hydrophilic residues predominantly on the opposite face when the
peptide is viewed along the helical axis.
[0173] Antimicrobial peptides of widely varying sequence have been
isolated, sharing an amphipathic .alpha.-helical structure as a
common feature (Saberwal et al., Biochim Biophys. Acta 1197:109-131
(1994)). Analogs of native peptides with amino acid substitutions
predicted to enhance amphipathicity and helicity typically have
increased antimicrobial activity. In general, analogs with
increased antimicrobial activity also have increased cytotoxicity
against mammalian cells (Maloy et al., Biopolymers 37:105-122
(1995)).
[0174] As used herein in reference to an antimicrobial peptide, the
term "amphipathic .alpha.-helical structure" means an .alpha.-helix
with a hydrophilic face containing several polar residues at
physiological pH and a hydrophobic face containing nonpolar
residues. A polar residue can be, for example, a lysine or arginine
residue, while a nonpolar residue can be, for example, a leucine or
alanine residue. An antimicrobial peptide having an amphipathic
.alpha.-helical structure generally has an equivalent number of
polar and nonpolar residues within the amphipathic domain and a
sufficient number of basic residues to give the peptide an overall
positive charge at neutral pH (Saberwal et al., Biochim Biophys.
Acta 1197:109-131 (1994)). One skilled in the art understands that
helix-promoting amino acids such as leucine and alanine can be
advantageously included in an antimicrobial peptide (see, for
example, Creighton, supra, 1984). Synthetic, antimicrobial peptides
having an amphipathic .alpha.-helical structure are known in the
art, for example, as described in U.S. Pat. No. 5,789,542 to
McLaughlin and Becker.
[0175] It is understood by one skilled in the art of medicinal
oncology that these and other agents are useful therapeutic agents,
which can be used separately or together in the disclosed
compositions and methods. Thus, it is understood that the
compositions disclosed herein can contain one or more of such
therapeutic agents and that additional components can be included
as part of the composition, if desired. As a non-limiting example,
it can be desirable in some cases to utilize an oligopeptide spacer
between the surface molecule and the homing molecule and/or cargo
molecules (Fitzpatrick and Garnett, Anticancer Drug Des. 10:1-9
(1995)).
[0176] Other useful agents include thrombolytics, aspirin,
anticoagulants, painkillers and tranquilizers, beta-blockers,
ace-inhibitors, nitrates, rhythm-stabilizing drugs, and diuretics.
Agents that limit damage to the heart work best if given within a
few hours of the heart attack. Thrombolytic agents that break up
blood clots and enable oxygen-rich blood to flow through the
blocked artery increase the patient's chance of survival if given
as soon as possible after the heart attack. Thrombolytics given
within a few hours after a heart attack are the most effective.
Injected intravenously, these include anisoylated plasminogen
streptokinase activator complex (APSAC) or anistreplase,
recombinant tissue-type plasminogen activator (r-tPA), and
streptokinase. The disclosed compounds can use any of these or
similar agents.
[0177] Some other examples of useful therapeutic agents include
nitrogen mustards, nitrosoureas, ethyleneimine, alkane sulfonates,
tetrazine, platinum compounds, pyrimidine analogs, purine analogs,
antimetabolites, folate analogs, anthracyclines, taxanes, vinca
alkaloids, topoisomerase inhibitors and hormonal agents. Exemplary
chemotherapy drugs are Actinomycin-D, Alkeran, Ara-C, Anastrozole,
Asparaginase, BiCNU, Bicalutamide, Bleomycin, Busulfan,
Capecitabine, Carboplatin, Carboplatinum, Carmustine, CCNU,
Chlorambucil, Chlomaphazine, Cholophosphamide, Cisplatin,
Cladribine, CPT-11, Cyclophosphamide, Cytarabine, Cytosine
arabinoside, Cytoxan, Dacarbazine, Dactinomycin, Daunorubicin,
Dexrazoxane, Docetaxel, Doxorubicin, DTIC, Epirubicin,
Estramustine, Ethyleneimine, Etoposide, Floxuridine, Fludarabine,
Fluorouracil, Flutamide, Fotemustine, Gemcitabine, Herceptin,
Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan,
Lomustine, Mechlorethamine, mechlorethamine oxide hydrochloride,
Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane,
Mitoxantrone, Novembiehin, Oxaliplatin, Paclitaxel, Pamidronate,
Pentostatin, Phenesterine, Plicamycin, Prednimustine, Procarbazine,
Rituximab, Steroids, Streptozocin, STI-571, Streptozocin,
Tamoxifen, Temozolomide, Teniposide, Tetrazine, Thioguanine,
Thiotepa, Tomudex, Topotecan, Treosulphan, Trimetrexate,
Trofosfamide, Vinblastine, Vincristine, Vindesine, Vinorelbine,
VP-16, and Xeloda. Alkylating agents such as Thiotepa and; alkyl
sulfonates such as Busulfan, Improsulfan and Piposulfan; aziridines
such as Benzodopa, Carboquone, Meturedopa, and Uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitroureas
such as Cannustine, Chlorozotocin, Fotemustine, Lomustine,
Nimustine, and Ranimustine; antibiotics such as Aclacinomysins,
Actinomycin, Authramycin, Azaserine, Bleomycins, Cactinomycin,
Calicheamicin, Carabicin, Caminomycin, Carzinophilin,
Chromoinycins, Dactinomycin, Daunorubicin, Detorubicin,
6-diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Esorubicin,
Idambicin, Marcellomycin, Mitomycins, mycophenolic acid,
Nogalamycin, Olivomycins, Peplomycin, Potfiromycin, Puromycin,
Quelamycin, Rodorubicin, Streptonigrin, Streptozocin, Tubercidin,
Ubenimex, Zinostatin, and Zorubicin; anti-metabolites such as
Methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as Denopterin, Methotrexate, Pteropterin, and Trimetrexate; purine
analogs such as Fludarabine, 6-mercaptopurine, Thiamiprine, and
Thioguanine; pyrimidine analogs such as Ancitabine, Azacitidine,
6-azauridine, Carmofur, Cytarabine, Dideoxyuridine, Doxifluridine,
Enocitabine, Floxuridine, and 5-FU; androgens such as Calusterone,
Dromostanolone Propionate, Epitiostanol, Rnepitiostane, and
Testolactone; anti-adrenals such as aminoglutethimide, Mitotane,
and Trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine;
Demecolcine; Diaziquone; Elfornithine; elliptinium acetate;
Etoglucid; gallium nitrate; hydroxyurea; Lentinan; Lonidamine;
Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin;
Phenamet; Pirarubicin; podophyllinic acid; 2-ethylhydrazide;
Procarbazine; PSK.RTM.; Razoxane; Sizofrran; Spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
Urethan; Vindesine; Dacarbazine; Mannomustine; Mitobronitol;
Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C");
cyclophosphamide; thiotEPa; taxoids, e.g., Paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and Doxetaxel
(TAXOTERE.RTM., Rhone-Poulenc Rorer, Antony, France); Gemcitabine;
6-thioguanine; Mercaptopurine; Methotrexate; platinum analogs such
as Cisplatin and Carboplatin; Vinblastine; platinum; etoposide
(VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine;
Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin;
Aminopterin; Xeloda; Ibandronate; CPT-11; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
Esperamicins; Capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included are
anti-hormonal agents that act to regulate or inhibit hormone action
on tumors such as anti-estrogens including for example Tamoxifen,
Raloxifene, aromatase inhibiting 4(5)-imidazoles, 4
Hydroxytamoxifen, Trioxifene, Keoxifene, Onapristone, And
Toremifene (Fareston); and anti-androgens such as Flutamide,
Nilutamide, Bicalutamide, Leuprolide, and Goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. Useful cargo molecules include, for example,
doxorubicin, Herceptin, and liposomal doxorubicin.
[0178] The cargo molecules can also comprise a boron containing
compound. Boron containing compounds have received increasing
attention as therapeutic agents over the past few years as
technology in organic synthesis has expanded to include this atom
(Boron Therapeutics on the horizon, Groziak, M. P.; American
Journal of Therapeutics (2001) 8, 321-328). The most notable boron
containing therapeutic is the boronic acid bortezomib which was
recently launched for the treatment of multiple myeloma. This
breakthrough demonstrates the feasibility of using boron containing
compounds as pharmaceutical agents. Boron containing compounds have
been shown to have various biological activities including
herbicides (Organic boron compounds as herbicides. Barnsley, G. E.;
Eaton, J. K.; Airs, R. S.; (1957), DE 1016978 19571003), boron
neutron capture therapy (Molecular Design and Synthesis of B-10
Carriers for Neutron Capture Therapy. Yamamoto, Y.; Pure Appl.
Chem., (1991) 63, 423-426), serine protease inhibition (Borinic
acid inhibitors as probes of the factors involved in binding at the
active sites of subtilisin Carlsberg and alpha-chymotrypsin.
Simpelkamp, J.; Jones, J. B.; Bioorganic & Medicinal Chemistry
Letters, (1992), 2(11), 1391-4; Design, Synthesis and Biological
Evaluation of Selective Boron-containing Thrombin Inhibitors.
Weinand, A.; Ehrhardt, C.; Metternich, R.; Tapparelli, C.;
Bioorganic and Medicinal Chemistry, (1999), 7, 1295-1307),
acetylcholinesterase inhibition (New, specific and reversible
bifunctional alkylborinic acid inhibitor of acetylcholinesterase.
Koehler, K. A.; Hess, G. P.; Biochemistry (1974), 13, 5345-50) and
as antibacterial agents (Boron-Containing Antibacterial Agents:
Effects on Growth and Morphology of Bacteria Under Various Culture
Conditions. Bailey, P. J.; Cousins, G.; Snow, G. A.; and White, A.
J.; Antimicrobial Agents and Chemotherapy, (1980), 17, 549-553).
The boron containing compounds with antibacterial activity can be
sub-divided into two main classes, the diazaborinines, which have
been known since the 1960's, and dithienylborinic acid complexes.
This latter class has been expanded to include many different
diarylborinic acid complexes with potent antibacterial activity
(Preparation of diarylborinic acid esters as DNA methyl transferase
inhibitors. Benkovic, S. J.; Shapiro, L.; Baker, S. J.; Wahnon, D.
C.; Wall, M.; Shier, V. K.; Scott, C. P.; Baboval, J.; PCT Int.
Appl. (2002), WO 2002044184). [0179] ii. Detectable Agents
[0180] The moiety in the disclosed compositions can also be a
detectable agent. A variety of detectable agents are useful in the
disclosed methods. As used herein, the term "detectable agent"
refers to any molecule which can be detected. Useful detectable
agents include compounds and molecules that can be administered in
vivo and subsequently detected. Detectable agents useful in the
disclosed compositions and methods include yet are not limited to
radiolabels and fluorescent molecules. The detectable agent can be,
for example, any molecule that facilitates detection, either
directly or indirectly, preferably by a non-invasive and/or in vivo
visualization technique. For example, a detectable agent can be
detectable by any known imaging techniques, including, for example,
a radiological technique, a magnetic resonance technique, or an
ultrasound technique. Detectable agents can include, for example, a
contrasting agent, e.g., where the contrasting agent is ionic or
non-ionic. In some embodiments, for instance, the detectable agent
comprises a tantalum compound and/or a barium compound, e.g.,
barium sulfate. In some embodiments, the detectable agent comprises
iodine, such as radioactive iodine. In some embodiments, for
instance, the detectable agent comprises an organic iodo acid, such
as iodo carboxylic acid, triiodophenol, iodoform, and/or
tetraiodoethylene. In some embodiments, the detectable agent
comprises a non-radioactive detectable agent, e.g., a
non-radioactive isotope. For example, Gd can be used as a
non-radioactive detectable agent in certain embodiments.
[0181] Other examples of detectable agents include molecules which
emit or can be caused to emit detectable radiation (e.g.,
fluorescence excitation, radioactive decay, spin resonance
excitation, etc.), molecules which affect local electromagnetic
fields (e.g., magnetic, ferromagnetic, ferromagnetic, paramagnetic,
and/or superparamagnetic species), molecules which absorb or
scatter radiation energy (e.g., chromophores and/or fluorophores),
quantum dots, heavy elements and/or compounds thereof. See, e.g.,
detectable agents described in U.S. Publication No. 2004/0009122.
Other examples of detectable agents include a proton-emitting
molecules, a radiopaque molecules, and/or a radioactive molecules,
such as a radionuclide like Tc-99m and/or Xe-13. Such molecules can
be used as a radiopharmaceutical. In still other embodiments, the
disclosed compositions can comprise one or more different types of
detectable agents, including any combination of the detectable
agents disclosed herein.
[0182] Useful fluorescent moieties include fluorescein
isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red,
nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,
rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin,
BODIPY.RTM., Cascade Blue.RTM., Oregon Green.RTM., pyrene,
lissamine, xanthenes, acridines, oxazines, phycoerythrin,
macrocyclic chelates of lanthanide ions such as quantum dye.TM.,
fluorescent energy transfer dyes, such as thiazole orange-ethidium
heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
Examples of other specific fluorescent labels include
3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine
(5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red,
Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon
Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon
Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G,
BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate,
Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy F1,
Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor
RW Solution, Calcofluor White, Calcophor White ABT Solution,
Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,
Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin,
CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic
Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH-CH3,
Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid,
Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF,
Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced
Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2,
Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl
Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue,
Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, Leucophor PAF,
Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),
Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue,
Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF,
MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine,
Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear
Yellow, Nylosan Brilliant Flavin E8G, Oxadiazole, Pacific Blue,
Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL,
Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine,
Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin,
Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant
Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD,
Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,
Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron
Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B,
Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene
Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can
C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine Red R,
Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol
Orange, Tinopol CBS, True Blue, Ultralite, Uranine B, Uvitex SFC,
Xylene Orange, and XRITC.
[0183] Particularly useful fluorescent labels include fluorescein
(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine
(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5,
Cy5.5 and Cy7. The absorption and emission maxima, respectively,
for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm),
Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703
nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous
detection. Other examples of fluorescein dyes include
6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein
(TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX),
2',7'-dimethoxy-4', 5'-dichloro-6-carboxyrhodamine (JOE),
2'-chloro-5'-fluoro-7',8'-fused
phenyl-1,4-dichloro-6-carboxyfluorescein (NED), and
2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).
Fluorescent labels can be obtained from a variety of commercial
sources, including Amersham Pharmacia Biotech, Piscataway, N.J.;
Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland,
Ohio. Fluorescent probes and there use are also described in
Handbook of Fluorescent Probes and Research Products by Richard P.
Haugland.
[0184] Further examples of radioactive detectable agents include
gamma emitters, e.g., the gamma emitters In-111, I-125 and I-131,
Rhenium-186 and 188, and Br-77 (see. e.g., Thakur, M. L. et al.,
Throm Res. Vol. 9 pg. 345 (1976); Powers et al., Neurology Vol. 32
pg. 938 (1982); and U.S. Pat. No. 5,011,686); positron emitters,
such as Cu-64, C-11, and O-15, as well as Co-57, Cu-67, Ga-67,
Ga-68, Ru-97, Tc-99m, In-113m, Hg-197, Au-198, and Pb-203. Other
radioactive detectable agents can include, for example tritium,
C-14 and/or thallium, as well as Rh-105, I-123, Nd-147, Pm-151,
Sm-153, Gd-159, Tb-161, Er-171 and/or Tl-201.
[0185] The use of Technitium-99m (Tc-99m) is preferable and has
been described in other applications, for example, see U.S. Pat.
No. 4,418,052 and U.S. Pat. No. 5,024,829. Tc-99m is a gamma
emitter with single photon energy of 140 keV and a half-life of
about 6 hours, and can readily be obtained from a Mo-99/Tc-99
generator.
[0186] In some embodiments, compositions comprising a radioactive
detectable agent can be prepared by coupling a targeting moiety
with radioisotopes suitable for detection. Coupling can occur via a
chelating agent such as diethylenetriaminepentaacetic acid (DTPA),
4,7,10-tetraazacyclododecane-N-,N',N'',N'''-tetraacetic acid (DOTA)
and/or metallothionein, any of which can be covalently attached to
the targeting moiety. In some embodiments, an aqueous mixture of
technetium-99m, a reducing agent, and a water-soluble ligand can be
prepared and then allowed to react with a disclosed targeting
moiety. Such methods are known in the art, see e.g., International
Publication No. WO 99/64446. In some embodiments, compositions
comprising radioactive iodine, can be prepared using an exchange
reaction. For example, exchange of hot iodine for cold iodine is
well known in the art. Alternatively, a radio-iodine labeled
compound can be prepared from the corresponding bromo compound via
a tributylstannyl intermediate.
[0187] Magnetic detectable agents include paramagnetic contrasting
agents, e.g., gadolinium diethylenetriaminepentaacetic acid, e.g.,
used with magnetic resonance imaging (MRI) (see, e.g., De Roos, A.
et al., Int. J. Card. Imaging Vol. 7 pg. 133 (1991)). Some
preferred embodiments use as the detectable agent paramagnetic
atoms that are divalent or trivalent ions of elements with an
atomic number 21, 22, 23, 24, 25, 26, 27, 28, 29, 42, 44, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. Suitable ions
include, but are not limited to, chromium(III), manganese(II),
iron(II), iron(III), cobalt(II), nickel(II), copper(II),
praseodymium(III), neodymium(III), samarium(III) and
ytterbium(III), as well as gadolinium(III), terbiurn(III),
dysoprosium(III), holmium(III), and erbium(III). Some preferred
embodiments use atoms with strong magnetic moments, e.g.,
gadolinium(III).
[0188] In some embodiments, compositions comprising magnetic
detectable agents can be prepared by coupling a targeting moiety
with a paramagnetic atom. For example, the metal oxide or a metal
salt, such as a nitrate, chloride or sulfate salt, of a suitable
paramagnetic atom can be dissolved or suspended in a water/alcohol
medium, such as methyl, ethyl, and/or isopropyl alcohol. The
mixture can be added to a solution of an equimolar amount of the
targeting moiety in a similar water/alcohol medium and stirred. The
mixture can be heated moderately until the reaction is complete or
nearly complete. Insoluble compositions formed can be obtained by
filtering, while soluble compositions can be obtained by
evaporating the solvent. If acid groups on the chelating moieties
remain in the disclosed compositions, inorganic bases (e.g.,
hydroxides, carbonates and/or bicarbonates of sodium, potassium
and/or lithium), organic bases, and/or basic amino acids can be
used to neutralize acidic groups, e.g., to facilitate isolation or
purification of the composition.
[0189] The detectable agent can be coupled to the composition in
such a way so as not to interfere with the ability of the homing
molecule to interact with the target site. In some embodiments, the
detectable agent can be chemically bound to, for example, the
surface molecule, homing molecule, and/or membrane perturbing
molecule. In some embodiments, the detectable agent can be
chemically bound to a moiety that is itself chemically bound to,
for example, the surface molecule, homing molecule, and/or membrane
perturbing molecule, indirectly linking the imaging and targeting
moieties.
C. Internalization Elements and Tissue Penetration Elements
[0190] The disclosed compositions, surface molecules, cargo
molecules, peptides, proteins, amino acid sequences, etc. can
comprise one or more internalization elements, tissue penetration
elements, or both. Internalization elements and tissue penetration
elements can be incorporated into or fused with other peptide
components of the composition, such as peptide homing molecules and
peptide cargo molecules. Internalization elements are molecules,
often peptides or amino acid sequences, that allow the
internalization element and components with which it is associated,
to pass through biological membranes. Tissue penetration elements
are molecules, often peptides or amino acid sequences, that allow
the tissue penetration element and components with which it is
associated to passage into and through tissue. "Internalization"
refers to passage through a plasma membrane or other biological
barrier. "Penetration" refers to passage into and through a
membrane, cell, tissue, or other biological barrier. Penetration
generally involves and includes internalization. Some molecules,
such as CendR elements, function as both internalization elements
and tissue penetration elements.
[0191] Internalization elements include, for example,
cell-penetrating peptides (CPPs) and CendR peptides. Peptides that
are internalized into cells are commonly referred to as
cell-penetrating peptides. There are two main classes of such
peptides: hydrophobic and cationic (Zorko and Langel, 2005). The
cationic peptides, which are commonly used to introduce nucleic
acids, proteins into cells, include the prototypic cell-penetrating
peptides (CPP), Tat, and penetratin (Derossi et al., 1998; Meade
and Dowdy, 2007). A herpes virus protein, VP22, is capable of both
entering and exiting cells and carrying a payload with it (Elliott
and O'Hare, 1997; Brewis et al., 2003).
[0192] 1. CendR Elements
[0193] Useful forms of internalization elements and tissue
penetration elements are CendR elements. CendR elements are amino
acid sequences with a C-terminal element as a defining feature that
signals highly efficient internalization of phage and free peptides
into cells. This internalization phenomenon has been named the
"C-end rule" or "CendR". The CendR pathway can also be used for
passage of compositions of interest from the vasculature and their
spread into tissue. The C-terminal element can cause spread of
compositions from the vasculature (and thus can be spread into
tumor tissue from an intravenous injection, for example). CendR
elements can also be used to mediate passage of compositions of
interest through other CendR-capable membranes, such as mucous
membranes and the blood-brain barrier. As used herein, "tissue
penetration" and "penetration of tissue" refer to passage into or
through a tissue beyond or through the outer or a first layer of
cells or through a tissue membrane. Such passage or penetration
through tissue (which can also be referred to as extravasation and
tissue penetration) can be a function of, for example, cell
internalization and passage between cells in the tissue. Throughout
this application, when the term "tissue penetration" is used, it is
understood that such penetration can also extend to other barriers
and CendR-capable membranes found throughout the body, such as the
blood brain barrier.
[0194] Unlike the known cell-penetrating peptides, the CendR
internalizing element is position-dependent--it is inactive when
present in positions other than the C-terminus of the peptide.
Another distinguishing feature is that the CendR element is
stereo-specific, that is, CendR elements composed of D-amino acids
are inactive. A latent CendR peptide can be activated by cleavage
by, for example, the appropriate proteolytic enzyme to expose, for
example, a C-terminal arginine, lysine, or lysine-glycine.
Throughout the application, when the term "CendR element" or
"C-terminal element" is used, it is used to describe a C-terminal
arginine, a C-terminal lysine, or a C-terminal lysine-glycine pair,
where glycine is at the furthest C-terminal position. In other
words, in the case where a lysine is on the C terminus end, the
CendR element can remain functional with a glycine on the C
terminus side of the lysine. However, it is not necessary to have
glycine on the end in order for the lysine residue to be functional
as a C-terminal element, so that lysine can be present without
glycine and still be functional. The converse is not true, however,
in that glycine cannot function as a C-terminal element without the
presence of lysine adjacent to it. Arginine does not require either
lysine or glycine to function as a C-terminal element, as long as
it remains in the furthest C-terminal position. Such CendR elements
can be referred to as type 1 CendR elements.
[0195] The term "CendR element" or "C-terminal element" can also be
used to describe a C-terminal histidine and amino acid sequences
having the sequence X.sub.1X.sub.2X.sub.3X.sub.4, where X.sub.1 can
be R, K or H, where X.sub.4 can be R, K, H, or KG, and where
X.sub.2 and X.sub.3 can each be, independently, any amino acid.
Such CendR elements can be referred to as type 2 CendR elements.
The X.sub.2 and X.sub.3 amino acids can be selected for specific
purposes. For example, X.sub.2, X.sub.3, or both can be chosen to
form all or a portion of a protease recognition sequence. This
would be useful, for example, to specify or enable cleavage of a
peptide having the CendR element as a latent or cryptic CendR
element that is activated by cleavage following the X.sub.4 amino
acid. Examples of such amino acid choices are shown in Tables 1 and
2. The X.sub.1, X.sub.2 and X.sub.3 amino acids can also be
selected, for example, to recruit additional proteins to NRP-1
molecules at the cell surface. This can be applied, for example, to
modulate the selectivity and internalization and/or tissue
penetration potency of CendR elements (and the compositions,
conjugates, proteins, and peptides containing CendR elements). The
X.sub.2 and X.sub.3 amino acids can also be selected to prevent
protease cleavage within the X.sub.1-X.sub.4 motif. Optionally,
certain amino acids can also be excluded from use for X.sub.2,
X.sub.3, or both. For example, if desired, G and D can be excluded
from simultaneous use as X.sub.2 and X.sub.3, respectively. Some
type 2 CendR elements can also be described as R/K/HXXR/K/H (SEQ ID
NO:130) and R/K/HXXKG (SEQ ID NO:131).
[0196] Examples of CendR elements include XXR/K/H, XXR/K, XXR/H,
XXK/H, XXR, XXK, XXH, XXKG, RXXR/K/H, RXXR/K, RXXR/H, RXXK/H, RXXR,
RXXK, RXXH, RXXKG, KXXR/K/H, KXXR/K, KXXR/H, KXXK/H, KXXR, KXXK,
KXXH, KXXKG, HXXR/K/H, HXXR/K, HXXR/H, HXXK/H, HXXR, HXXK, HXXH,
HXXKG, R/K/HXXR, R/KXXR, R/HXXR, K/HXXR, RXXR, KXXR, HXXR,
R/K/HXXK, R/KXXK, R/HXXK, K/HXXK, RXXK, KXXK, HXXK, R/K/HXXH,
R/KXXH, R/HXXH, K/HXXH, RXXH, KXXH, HXXH, R/K/HXXKG, R/KXXKG,
R/HXXKG, K/HXXKG, RXXKG, KXXKG, and HXXKG.
[0197] A CendR element that can be internalized into a cell can be
referred to as an internalization CendR element. A CendR element
that can penetrate tissue can be referred to as a penetrating CendR
element. A CendR element that can be internalized into a cell and
that can penetrate tissue can be referred to as an internalization
and penetrating CendR element. Unless the context clearly indicates
otherwise, reference to "CendR element" refers to any of these,
either individually, collectively, or in any combination.
[0198] As used herein, "CendR composition" refers to a composition
that comprises a CendR element. The CendR element can be, for
example, active, activatable, or blocked. For example, the CendR
composition can comprise a protein or peptide comprising an amino
acid sequence that comprises a CendR element where the amino acid
sequence is at the C-terminal end of the protein or peptide.
[0199] As used herein, "activatable CendR element" refers to a
CendR element having a molecule, moiety, nanoparticle, compound or
other composition covalently coupled to the CendR element, such as
to the terminal carboxyl group of the C-terminal element, where the
molecule, moiety, nanoparticle, compound or other composition can
block internalization and/or tissue penetration of the CendR
composition, conjugate, molecule, protein, peptide, etc. and where
the molecule, moiety, nanoparticle, compound or other composition
can be removed (to expose the terminal carboxy group, for example).
For example, the activatable CendR element can be on the C-terminal
end of the peptide, and can prevent the CendR element from being
internalized and/or from penetrating tissue. The molecule,
nanoparticle, moiety, compound or other composition covalently
coupled to the CendR element can be referred to as the "blocking
group." For example, the blocking group can be coupled to the
terminal carboxyl group of the C-terminal arginine or lysine or
other C-terminal amino acid of the CendR element, to the C-terminal
amino acid of the CendR element, or to an amino acid of the CendR
element other than the C-terminal amino acid. The blocking group
can also be coupled, or associated with a part of a CendR
composition, conjugate, molecule, protein, peptide, etc. other than
the CendR element so long as it can prevent the CendR element from
being internalized and/or from penetrating tissue. A CendR
composition comprising an activatable CendR element can be referred
to as an activatable CendR composition. A CendR molecule comprising
an activatable CendR element can be referred to as an activatable
CendR molecule. A CendR conjugate comprising an activatable CendR
element can be referred to as an activatable CendR conjugate. A
CendR protein comprising an activatable CendR element can be
referred to as an activatable CendR protein. A CendR peptide
comprising an activatable CendR element can be referred to as an
activatable CendR peptide.
[0200] An activatable CendR element can be blocked from
internalization into a cell, from tissue penetration, or both.
Generally, an activatable CendR element will be blocked from both
internalization into a cell and penetration of tissue. Such
activatable CendR elements can be referred to as activatable
internalization and penetrating CendR elements. However, some
activatable CendR elements could be blocked only from tissue
penetration or only from internalization into a cell. Such
activatable CendR elements can be referred to as activatable
internalization CendR elements (for CendR elements that are blocked
only from internalization into a cell) or as activatable
internalization and penetrating CendR elements (for CendR elements
that are blocked only from penetration of tissue). Generally,
internalization CendR elements that are activatable will be
activatable internalization CendR elements. Similarly, penetrating
CendR elements that are activatable generally will be activatable
penetrating CendR elements. Internalization and penetrating CendR
elements that are activatable will be activatable internalization
and penetrating CendR elements. Removal of the blocking group will
allow the CendR element to be internalized into a cell, penetrate
tissue, or both.
[0201] The cleavable bond of an activatable CendR element can be
cleaved in any suitable way. For example, the cleavable bond can be
cleaved enzymatically or non-enzymatically. For enzymatic cleavage,
the cleaving enzyme can be supplied or can be present at a site
where the CendR element is delivered, homes, travels or
accumulates. For example, the enzyme can be present in proximity to
a cell to which the CendR element is delivered, homes, travels, or
accumulates. For non-enzymatic cleavage, the CendR element can be
brought into contact with a cleaving agent, can be placed in
cleaving conditions, or both. A cleaving agent is any substance
that can mediate or stimulate cleavage of the cleavable bond. A
non-enzymatic cleaving agent is any cleaving agent except enzymes.
Cleaving conditions can be any solution or environmental conditions
that can mediate or stimulate cleavage of the cleavable bond. For
example, some labile bonds can be cleaved in acid conditions,
alkaline conditions, in the presence of a reactive group, etc.
Non-enzymatic cleaving conditions are any cleaving conditions
except the presence of enzymes. Non-agent cleaving conditions are
any cleaving conditions except the presence of cleaving agents.
[0202] A "protease-activatable CendR element" (or
"protease-activated CendR element") refers to an activatable CendR
element where the blocking group is coupled to the CendR element
via a peptide bond and where the peptide bond can be cleaved by a
protease. Cleavage of this peptide bond in a protease-activatable
CendR element makes the CendR element capable of internalization
into a cell and/or of tissue penetration. In one example, the
blocking group can be coupled to the CendR element via a cleavable
or labile bond. The cleavable bond can be cleaved by, for example,
an enzyme or a chemical compound. Cleavage or `labilization` bond
in an activatable CendR element makes the CendR element capable of
internalization into a cell and/or of tissue penetration. Such
cleavage or `labilization` can be referred to as activation of the
CendR element. A protease-activatable CendR element is a form of
activatable CendR element.
[0203] Proteolysis that uncovers a C-terminal element can serve as
a switch that triggers the internalization signal. Various
compositions can be internalized through this mechanism. For
example, homing molecule-mediated accumulation can occur at a
target site with cell type-specific proteolysis that exposes a
C-terminal element which allows for highly specific homing systems
with target-triggered internalization. This protease-controllable
internalization system can be useful in engineering compositions
with functions such as cell type-specific and/or tissue
type-specific uptake and the ability to spread the compositions in
tissues.
[0204] CendR elements are further described in U.S. Patent
Application Publication Nos. 2009-0226372 and 2010-0322862, which
are hereby incorporated by reference in their entirety, and
specifically for their description of the form, structure, and use
of CendR elements and peptides.
D. Surface Molecules
[0205] The surface molecules, alternatively referred to as a
surface particles, disclosed herein can be conjugated with homing
molecules and cargo molecules in such a way that the composition is
delivered to a target. The surface molecule can be any substance
that can be used with the homing molecules and cargo molecules, and
is not restricted by size or substance. Examples include, but are
not limited to, nanoparticles (such as iron oxide nanoparticles or
albumin nanoparticles), liposomes, small organic molecules,
microparticles, or microbubbles, such as fluorocarbon microbubbles.
The term surface molecule is used to identify a component of the
disclosed composition but is not intended to be limiting. In
particular, the disclosed surface molecules are not limited to
substances, compounds, compositions, particles or other materials
composed of a single molecule. Rather, the disclosed surface
molecules are any substance(s), compound(s), composition(s),
particle(s) and/or other material(s) that can be conjugated with a
plurality of homing molecules and cargo molecules such that at
least some of the homing molecules and/or cargo molecules are
presented and/or accessible on the surface of the surface molecule.
A variety of examples of suitable surface molecules are described
and disclosed herein.
[0206] The surface molecule can be detectable, or can be a
therapeutic agent such as iRGD, RGD, or Abraxane.TM.. The section
herein which discusses cargo molecules and moieties that can be
detectable or therapeutic also applies to the surface molecule.
[0207] Surface molecules can be associated with and arranged in the
compositions in a variety of configurations. In some forms, surface
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of homing molecules, a plurality of cargo
molecules, or both. In some forms, surface molecules can be
associated with, conjugated to, and/or covalently coupled to a
plurality of homing molecules, wherein the homing molecules can be
associated with, conjugated to, and/or covalently coupled to a
plurality of cargo molecules. In some forms, surface molecules can
be associated with, conjugated to, and/or covalently coupled to a
plurality of cargo molecules, wherein the cargo molecules can be
associated with, conjugated to, and/or covalently coupled to a
plurality of homing molecules. Combinations of these combinations
can also be used.
[0208] 1. Nanoparticles, Microparticles, and Microbubbles
[0209] The term "nanoparticle" refers to a nanoscale particle with
a size that is measured in nanometers, for example, a nanoscopic
particle that has at least one dimension of less than about 100 nm.
Examples of nanoparticles include paramagnetic nanoparticles,
superparamagnetic nanoparticles, metal nanoparticles, nanoworms,
fullerene-like materials, inorganic nanotubes, dendrimers (such as
with covalently attached metal chelates), nanofibers, nanohoms,
nano-onions, nanorods, nanoropes and quantum dots. A nanoparticle
can produce a detectable signal, for example, through absorption
and/or emission of photons (including radio frequency and visible
photons) and plasmon resonance.
[0210] Microspheres (or microbubbles) can also be used with the
methods disclosed herein. Microspheres containing chromophores have
been utilized in an extensive variety of applications, including
photonic crystals, biological labeling, and flow visualization in
microfluidic channels. See, for example, Y. Lin, et al., Appl. Phys
Lett. 2002, 81, 3134; D. Wang, et al., Chem. Mater. 2003, 15, 2724;
X. Gao, et al., J. Biomed. Opt. 2002, 7, 532; M. Han, et al.,
Nature Biotechnology. 2001, 19, 631; V. M. Pai, et al., Mag. &
Magnetic Mater. 1999, 194, 262, each of which is incorporated by
reference in its entirety. Both the photostability of the
chromophores and the monodispersity of the microspheres can be
important.
[0211] Nanoparticles, such as, for example, metal nanoparticles,
metal oxide nanoparticles, or semiconductor nanocrystals can be
incorporated into microspheres. The optical, magnetic, and
electronic properties of the nanoparticles can allow them to be
observed while associated with the microspheres and can allow the
microspheres to be identified and spatially monitored. For example,
the high photostability, good fluorescence efficiency and wide
emission tunability of colloidally synthesized semiconductor
nanocrystals can make them an excellent choice of chromophore.
Unlike organic dyes, nanocrystals that emit different colors (i.e.
different wavelengths) can be excited simultaneously with a single
light source. Colloidally synthesized semiconductor nanocrystals
(such as, for example, core-shell CdSe/ZnS and CdS/ZnS
nanocrystals) can be incorporated into microspheres. The
microspheres can be monodisperse silica microspheres.
[0212] The nanoparticle can be a metal nanoparticle, a metal oxide
nanoparticle, or a semiconductor nanocrystal. The metal of the
metal nanoparticle or the metal oxide nanoparticle can include
titanium, zirconium, hafnium, vanadium, niobium, tantalum,
chromium, molybdenum, tungsten, manganese, technetium, rhenium,
iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel,
palladium, platinum, copper, silver, gold, zinc, cadmium, scandium,
yttrium, lanthanum, a lanthanide series or actinide series element
(e.g., cerium, praseodymium, neodymium, promethium, samarium,
europium, gadolinium, terbium, dysprosium, holmium, erbium,
thulium, ytterbium, lutetium, thorium, protactinium, and uranium),
boron, aluminum, gallium, indium, thallium, silicon, germanium,
tin, lead, antimony, bismuth, polonium, magnesium, calcium,
strontium, and barium. In certain embodiments, the metal can be
iron, ruthenium, cobalt, rhodium, nickel, palladium, platinum,
silver, gold, cerium or samarium. The metal oxide can be an oxide
of any of these materials or combination of materials. For example,
the metal can be gold, or the metal oxide can be an iron oxide, a
cobalt oxide, a zinc oxide, a cerium oxide, or a titanium oxide.
Preparation of metal and metal oxide nanoparticles is described,
for example, in U.S. Pat. Nos. 5,897,945 and 6,759,199, each of
which is incorporated by reference in its entirety.
[0213] The nanoparticles can be comprised of cargo molecules and a
carrier protein (such as albumin) Such nanoparticles are useful,
for example, to deliver hydrophobic or poorly soluble compounds.
Nanoparticles of poorly water soluble drugs (such as taxane) have
been disclosed in, for example, U.S. Pat. Nos. 5,916,596;
6,506,405; and 6,537,579 and also in U.S. Pat. Pub. No.
2005/0004002A1.
[0214] In forms, the nanoparticles can have an average or mean
diameter of no greater than about 1000 nanometers (nm), such as no
greater than about any of 900, 800, 700, 600, 500, 400, 300, 200,
and 100 nm. In some forms, the average or mean diameters of the
nanoparticles can be no greater than about 200 nm. In some forms,
the average or mean diameters of the nanoparticles can be no
greater than about 150 nm. In some forms, the average or mean
diameters of the nanoparticles can be no greater than about 100 nm.
In some forms, the average or mean diameter of the nanoparticles
can be about 20 to about 400 nm. In some forms, the average or mean
diameter of the nanoparticles can be about 40 to about 200 nm. In
some embodiments, the nanoparticles are sterile-filterable.
[0215] The nanoparticles can be present in a dry formulation (such
as lyophilized composition) or suspended in a biocompatible medium.
Suitable biocompatible media include, but are not limited to,
water, buffered aqueous media, saline, buffered saline, optionally
buffered solutions of amino acids, optionally buffered solutions of
proteins, optionally buffered solutions of sugars, optionally
buffered solutions of vitamins, optionally buffered solutions of
synthetic polymers, lipid-containing emulsions, and the like.
[0216] Examples of suitable carrier proteins include proteins
normally found in blood or plasma, which include, but are not
limited to, albumin, immunoglobulin including IgA, lipoproteins,
apolipoprotein B, alpha-acid glycoprotein, beta-2-macroglobulin,
thyroglobulin, transferin, fibronectin, factor VII, factor VIII,
factor IX, factor X, and the like. In some embodiments, the carrier
protein is non-blood protein, such as casein, .alpha.-lactalbumin,
and .beta.-lactoglobulin. The carrier proteins may either be
natural in origin or synthetically prepared. In some embodiments,
the pharmaceutically acceptable carrier comprises albumin, such as
human serum albumin Human serum albumin (HSA) is a highly soluble
globular protein of M.sub.r 65K and consists of 585 amino acids.
HSA is the most abundant protein in the plasma and accounts for
70-80% of the colloid osmotic pressure of human plasma. The amino
acid sequence of HSA contains a total of 17 disulphide bridges, one
free thiol (Cys 34), and a single tryptophan (Trp 214). Intravenous
use of HSA solution has been indicated for the prevention and
treatment of hypovolumic shock (see, e.g., Tullis, JAMA
237:355-360, 460-463 (1977)) and Houser et al., Surgery, Gynecology
and Obstetrics, 150:811-816 (1980)) and in conjunction with
exchange transfusion in the treatment of neonatal
hyperbilirubinemia (see, e.g., Finlayson, Seminars in Thrombosis
and Hemostasis, 6:85-120 (1980)). Other albumins are contemplated,
such as bovine serum albumin. Use of such non-human albumins could
be appropriate, for example, in the context of use of these
compositions in non-human mammals, such as the veterinary
(including domestic pets and agricultural context).
[0217] Carrier proteins (such as albumin) in the composition
generally serve as a carrier for the hydrophobic cargo molecules,
i.e., the carrier protein in the composition makes the cargo
molecules more readily suspendable in an aqueous medium or helps
maintain the suspension as compared to compositions not comprising
a carrier protein. This can avoid the use of toxic solvents (or
surfactants) for solubilizing the cargo molecules, and thereby can
reduce one or more side effects of administration of the cargo
molecules into an individual (such as a human). Thus, in some
embodiments, the composition described herein can be substantially
free (such as free) of surfactants, such as Cremophor (including
Cremophor EL.RTM. (BASF)). In some embodiments, the composition can
be substantially free (such as free) of surfactants. A composition
is "substantially free of Cremophor" or "substantially free of
surfactant" if the amount of Cremophor or surfactant in the
composition is not sufficient to cause one or more side effect(s)
in an individual when the composition is administered to the
individual.
[0218] The amount of carrier protein in the composition described
herein will vary depending on other components in the composition.
In some embodiments, the composition comprises a carrier protein in
an amount that is sufficient to stabilize the cargo molecules in an
aqueous suspension, for example, in the form of a stable colloidal
suspension (such as a stable suspension of nanoparticles). In some
embodiments, the carrier protein is in an amount that reduces the
sedimentation rate of the cargo molecules in an aqueous medium. For
particle-containing compositions, the amount of the carrier protein
also depends on the size and density of nanoparticles of the cargo
molecules.
[0219] Methods of making nanoparticle compositions are known in the
art. For example, nanoparticles containing cargo molecules and
carrier protein (such as albumin) can be prepared under conditions
of high shear forces (e.g., sonication, high pressure
homogenization, or the like). These methods are disclosed in, for
example, U.S. Pat. Nos. 5,916,596; 6,506,405; and 6,537,579 and
also in U.S. Pat. Pub. No. 2005/0004002A1.
[0220] Briefly, the hydrophobic carrier molecules can be dissolved
in an organic solvent, and the solution can be added to a human
serum albumin solution. The mixture is subjected to high pressure
homogenization. The organic solvent can then be removed by
evaporation. The dispersion obtained can be further lyophilized
Suitable organic solvent include, for example, ketones, esters,
ethers, chlorinated solvents, and other solvents known in the art.
For example, the organic solvent can be methylene chloride and
chloroform/ethanol (for example with a ratio of 1:9, 1:8, 1:7, 1:6,
1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or
9:1).
[0221] The nanoparticle can also be, for example, a heat generating
nanoshell. As used herein, "nanoshell" is a nanoparticle having a
discrete dielectric or semi-conducting core section surrounded by
one or more conducting shell layers. U.S. Pat. No. 6,530,944 is
hereby incorporated by reference herein in its entirety for its
teaching of the methods of making and using metal nanoshells.
Targeting molecules can be attached to the disclosed compositions
and/or carriers. For example, the targeting molecules can be
antibodies or fragments thereof, ligands for specific receptors, or
other proteins specifically binding to the surface of the cells to
be targeted.
[0222] 2. Liposomes
[0223] "Liposome" as the term is used herein refers to a structure
comprising an outer lipid bi- or multi-layer membrane surrounding
an internal aqueous space. Liposomes can be used to package any
biologically active agent for delivery to cells.
[0224] Materials and procedures for forming liposomes are
well-known to those skilled in the art. Upon dispersion in an
appropriate medium, a wide variety of phospholipids swell, hydrate
and form multilamellar concentric bilayer vesicles with layers of
aqueous media separating the lipid bilayers. These systems are
referred to as multilamellar liposomes or multilamellar lipid
vesicles ("MLVs") and have diameters within the range of 10 nm to
100 .mu.m. These MLVs were first described by Bangham, et al., J
Mol. Biol. 13:238-252 (1965). In general, lipids or lipophilic
substances are dissolved in an organic solvent. When the solvent is
removed, such as under vacuum by rotary evaporation, the lipid
residue forms a film on the wall of the container. An aqueous
solution that typically contains electrolytes or hydrophilic
biologically active materials is then added to the film. Large MLVs
are produced upon agitation. When smaller MLVs are desired, the
larger vesicles are subjected to sonication, sequential filtration
through filters with decreasing pore size or reduced by other forms
of mechanical shearing. There are also techniques by which MLVs can
be reduced both in size and in number of lamellae, for example, by
pressurized extrusion (Barenholz, et al., FEBS Lett. 99:210-214
(1979)).
[0225] Liposomes can also take the form of unilamnellar vesicles,
which are prepared by more extensive sonication of MLVs, and
consist of a single spherical lipid bilayer surrounding an aqueous
solution. Unilamellar vesicles ("ULVs") can be small, having
diameters within the range of 20 to 200 nm, while larger ULVs can
have diameters within the range of 200 nm to 2 .mu.m. There are
several well-known techniques for making unilamellar vesicles. In
Papahadjopoulos, et al., Biochim et Biophys Acta 135:624-238
(1968), sonication of an aqueous dispersion of phospholipids
produces small ULVs having a lipid bilayer surrounding an aqueous
solution. Schneider, U.S. Pat. No. 4,089,801 describes the
formation of liposome precursors by ultrasonication, followed by
the addition of an aqueous medium containing amphiphilic compounds
and centrifugation to form a biomolecular lipid layer system.
[0226] Small ULVs can also be prepared by the ethanol injection
technique described by Batzri, et al., Biochim et Biophys Acta
298:1015-1019 (1973) and the ether injection technique of Deamer,
et al., Biochim et Biophys Acta 443:629-634 (1976). These methods
involve the rapid injection of an organic solution of lipids into a
buffer solution, which results in the rapid formation of
unilamellar liposomes. Another technique for making ULVs is taught
by Weder, et al. in "Liposome Technology", ed. G. Gregoriadis, CRC
Press Inc., Boca Raton, Fla., Vol. I, Chapter 7, pg. 79-107 (1984).
This detergent removal method involves solubilizing the lipids and
additives with detergents by agitation or sonication to produce the
desired vesicles.
[0227] Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes
the preparation of large ULVs by a reverse phase evaporation
technique that involves the formation of a water-in-oil emulsion of
lipids in an organic solvent and the drug to be encapsulated in an
aqueous buffer solution. The organic solvent is removed under
pressure to yield a mixture which, upon agitation or dispersion in
an aqueous media, is converted to large ULVs. Suzuki et al., U.S.
Pat. No. 4,016,100, describes another method of encapsulating
agents in unilamellar vesicles by freezing/thawing an aqueous
phospholipid dispersion of the agent and lipids.
[0228] In addition to the MLVs and ULVs, liposomes can also be
multivesicular. Described in Kim, et al., Biochim et Biophys Acta
728:339-348 (1983), these multivesicular liposomes are spherical
and contain internal granular structures. The outer membrane is a
lipid bilayer and the internal region contains small compartments
separated by bilayer septum. Still yet another type of liposomes
are oligolamellar vesicles ("OLVs"), which have a large center
compartment surrounded by several peripheral lipid layers. These
vesicles, having a diameter of 2-15 .mu.m, are described in Callo,
et al., Cryobiology 22(3):251-267 (1985).
[0229] Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also
describe methods of preparing lipid vesicles. More recently, Hsu,
U.S. Pat. No. 5,653,996 describes a method of preparing liposomes
utilizing aerosolization and Yiournas, et al., U.S. Pat. No.
5,013,497 describes a method for preparing liposomes utilizing a
high velocity-shear mixing chamber. Methods are also described that
use specific starting materials to produce ULVs (Wallach, et al.,
U.S. Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848
and 5,628,936).
[0230] A comprehensive review of all the aforementioned lipid
vesicles and methods for their preparation are described in
"Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca
Raton, Fla., Vol. I, II & III (1984). This and the
aforementioned references describing various lipid vesicles
suitable for use in the invention are incorporated herein by
reference.
[0231] 3. Micelles
[0232] "Micelle" as used herein refers to a structure comprising an
outer lipid monolayer. Micelles can be formed in an aqueous medium
when the Critical Micelle Concentration (CMC) is exceeded. Small
micelles in dilute solution at approximately the critical micelle
concentration (CMC) are generally believed to be spherical.
However, under other conditions, they may be in the shape of
distorted spheres, disks, rods, lamellae, and the like. Micelles
formed from relatively low molecular weight amphiphile molecules
can have a high CMC so that the formed micelles dissociate rather
rapidly upon dilution. If this is undesired, amphiphile molecules
with large hydrophobic regions can be used. For example, lipids
with a long fatty acid chain or two fatty acid chains, such as
phospholipids and sphingolipids, or polymers, specifically block
copolymers, can be used.
[0233] Polymeric micelles have been prepared that exhibit CMCs as
low as 10.sup.-6 M (molar). Thus, they tend to be very stable while
at the same time showing the same beneficial characteristics as
amphiphile micelles. Any micelle-forming polymer presently known in
the art or as such may become known in the future may be used in
the disclosed compositions and methods. Examples of micelle-forming
polymers include, without limitation, methoxy poly(ethylene
glycol)-b-poly(.epsilon.-caprolactone), conjugates of poly(ethylene
glycol) with phosphatidyl-ethanolamine, poly(ethylene
glycol)-b-polyesters, poly(ethylene glycol)-b-poly(L-aminoacids),
poly(N-vinylpyrrolidone)-b1-poly(orthoesters),
poly(N-vinylpyrrolidone)-b-polyanhydrides and
poly(N-vinylpyrrolidone)-b-poly(alkyl acrylates).
[0234] Micelles can be produced by processes conventional in the
art. Examples of such are described in, for example, Liggins
(Liggins, R. T. and Burt, H. M., "Polyether-polyester diblock
copolymers for the preparation of paclitaxel loaded polymeric
micelle formulations." Adv. Drug Del. Rev. 54: 191-202, (2002));
Zhang, et al. (Zhang, X. et al., "Development of amphiphilic
diblock copolymers as micellar carriers of taxol." Int. J. Pharm.
132: 195-206, (1996)); and Churchill (Churchill, J. R., and
Hutchinson, F. G., "Biodegradable amphipathic copolymers." U.S.
Pat. No. 4,745,160, (1988)). In one such method,
polyether-polyester block copolymers, which are amphipathic
polymers having hydrophilic (polyether) and hydrophobic (polyester)
segments, are used as micelle forming carriers.
[0235] Another type of micelle can be formed using, for example,
AB-type block copolymers having both hydrophilic and hydrophobic
segments, as described in, for example, Tuzar (Tuzar, Z. and
Kratochvil, P., "Block and graft copolymer micelles in solution.",
Adv. Colloid Interface Sci. 6:201-232, (1976)); and Wilhelm, et al.
(Wilhelm, M. et al., "Poly(styrene-ethylene oxide) block copolymer
micelle formation in water: a fluorescence probe study.",
Macromolecules 24: 1033-1040 (1991)). These polymeric micelles are
able to maintain satisfactory aqueous stability. These micelles, in
the range of approximately <200 nm in size, are effective in
reducing non-selective RES scavenging and show enhanced
permeability and retention.
[0236] Further, U.S. Pat. No. 5,929,177 to Kataoka, et al.
describes a polymeric molecule which is usable as, inter alia, a
drug delivery carrier. The micelle is formed from a block copolymer
having functional groups on both of its ends and which comprises
hydrophilic/hydrophobic segments. The polymer functional groups on
the ends of the block copolymer include amino, carboxyl and
mercapto groups on the .alpha.-terminal and hydroxyl, carboxyl
group, aldehyde group and vinyl group on the .omega.-terminal. The
hydrophilic segment comprises polyethylene oxide, while the
hydrophobic segment is derived from lactide, lactone or
(meth)acrylic acid ester.
[0237] Further, for example,
poly(D,L-lactide)-b-methoxypolyethylene glycol (MePEG:PDLLA)
diblock copolymers can be made using MePEG 1900 and 5000. The
reaction can be allowed to proceed for 3 hr at 160.degree. C.,
using stannous octoate (0.25%) as a catalyst. However, a
temperature as low as 130.degree. C. can be used if the reaction is
allowed to proceed for about 6 hr, or a temperature as high as
190.degree. C. can be used if the reaction is carried out for only
about 2 hr.
[0238] As another example, N-isopropylacrylamide ("IPAAm") (Kohjin,
Tokyo, Japan) and dimethylacrylamide ("DMAAm") (Wako Pure
Chemicals, Tokyo, Japan) can be used to make hydroxyl-terminated
poly(IPAAm-co-DMAAm) in a radical polymerization process, using the
method of Kohori, F. et al. (1998). (Kohori, F. et al.,
"Preparation and characterization of thermally Responsive block
copolymer micelles comprising
poly(N-isopropylacrylamide-b-D,L-lactide)." J. Control. Rel. 55:
87-98, (1998)). The obtained copolymer can be dissolved in cold
water and filtered through two ultrafiltration membranes with a
10,000 and 20,000 molecular weight cut-off. The polymer solution is
first filtered through a 20,000 molecular weight cut-off membrane.
Then the filtrate was filtered again through a 10,000 molecular
weight cut-off membrane. Three molecular weight fractions can be
obtained as a result, a low molecular weight, a middle molecular
weight, and a high molecular weight fraction. A block copolymer can
then be synthesized by a ring opening polymerization of D,L-lactide
from the terminal hydroxyl group of the poly(IPAAm-co-DMAAm) of the
middle molecular weight fraction. The resulting
poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) copolymer can be purified
as described in Kohori, F. et al. (1999). (Kohori, F. et al.,
"Control of adriamycin cytotoxic activity using thermally
responsive polymeric micelles composed of
poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide)-
.-", Colloids Surfaces B: Biointerfaces 16: 195-205, (1999)).
[0239] Examples of block copolymers from which micelles can be
prepared which can be used to coat a support surface are found in
U.S. Pat. No. 5,925,720, to Kataoka, et al., U.S. Pat. No.
5,412,072 to Sakarai, et al., U.S. Pat. No. 5,410,016 to Kataoka,
et al., U.S. Pat. No. 5,929,177 to Kataoka, et al., U.S. Pat. No.
5,693,751 to Sakurai, et al., U.S. Pat. No. 5,449,513 to Yokoyama,
et al., WO 96/32434, WO 96/33233 and WO 97/0623, the contents of
all of which are incorporated by reference. Modifications thereof
which are prepared by introducing thereon a suitable functional
group (including an ethyleneically unsaturated polymerizable group)
are also examples of block copolymers from which micelles of the
present invention are preferably prepared. Preferable block
copolymers are those disclosed in the above-mentioned patents and
or international patent publications. If the block copolymer has a
sugar residue on one end of the hydrophilic polymer segment, as in
the block copolymer of WO 96/32434, the sugar residue should
preferably be subjected to Malaprade oxidation so that a
corresponding aldehyde group may be formed.
[0240] 4. Lipids
[0241] Lipids are synthetically or naturally-occurring molecules
which includes fats, waxes, sterols, prenol lipids, fat-soluble
vitamins (such as vitamins A, D, E and K), glycerolipids,
monoglycerides, diglycerides, triglycerides, glycerophospholipids,
sphingolipids, phospholipids, fatty acids monoglycerides,
saccharolipids and others. Lipids can be hydrophobic or amphiphilic
small molecules; the amphiphilic nature of some lipids allows them
to form structures such as monolayers, vesicles, micelles,
liposomes, bi-layers or membranes in an appropriate environment
i.e. aqueous environment. Any of a number of lipids can be used as
amphiphile molecules, including amphipathic, neutral, cationic, and
anionic lipids. Such lipids can be used alone or in combination,
and can also include bilayer stabilizing components such as
polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017, "Polyamide
Oligomers", by Ansell), peptides, proteins, detergents,
lipid-derivatives, such as PEG coupled to phosphatidylethanolamine
and PEG conjugated to ceramides (see, U.S. Pat. No. 5,885,613). In
a preferred embodiment, cloaking agents, which reduce elimination
of liposomes by the host immune system, can also be included, such
as polyamide-oligomer conjugates, e.g., ATTA-lipids, (see, U.S.
patent application Ser. No. 08/996,783, filed Feb. 2, 1998) and
PEG-lipid conjugates (see, U.S. Pat. Nos. 5,820,873, 5,534,499 and
5,885,613).
[0242] Any of a number of neutral lipids can be included, referring
to any of a number of lipid species which exist either in an
uncharged or neutral zwitterionic form at physiological pH,
including diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,
cholesterol, cerebrosides, and diacylglycerols.
[0243] Cationic lipids, carry a net positive charge at
physiological pH, can readily be used as amphiphile molecules. Such
lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy) propyl-N,N-N-triethylammonium chloride
("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP");
3.beta.-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Cho1"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
-ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl
carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine
("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"). Additionally, a number of commercial
preparations of cationic lipids can be used, such as LIPOFECTIN
(including DOTMA and DOPE, available from GIBCO/BRL), LIPOFECTAMINE
(comprising DOSPA and DOPE, available from GIBCO/BRL), and
TRANSFECTAM (comprising DOGS, in ethanol, from Promega Corp.).
[0244] Anionic lipids can be used as amphiphile molecules and
include, but are not limited to, phosphatidylglycerol, cardiolipin,
diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl
phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,
N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and
other anionic modifying groups joined to neutral lipids.
[0245] Amphiphatic lipids can also be suitable amphiphile
molecules. "Amphipathic lipids" refer to any suitable material,
wherein the hydrophobic portion of the lipid material orients into
a hydrophobic phase, while the hydrophilic portion orients toward
the aqueous phase. Such compounds include, but are not limited to,
fatty acids, phospholipids, aminolipids, and sphingolipids.
Representative phospholipids include sphingomyelin,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatdylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or
dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds,
such as sphingolipids, glycosphingolipid families, diacylglycerols,
and .beta.-acyloxyacids, can also be used. Additionally, such
amphipathic lipids can be readily mixed with other lipids, such as
triglycerides and sterols. Zwitterionic lipids are a form of
amphiphatic lipid.
[0246] Sphingolipids are fatty acids conjugated to the aliphatic
amino alcohol sphingosine. The fatty acid can be covalently bond to
sphingosine via an amide bond. Any amino acid as described above
can be covalently bond to sphingosine to form a sphingolipid. A
sphingolipid can be further modified by covalent bonding through
the .alpha.-hydroxyl group. The modification can include alkyl
groups, alkenyl groups, alkynyl groups, aromatic groups,
heteroaromatic groups, cyclyl groups, heterocyclyl groups,
phosphonic acid groups. Non-limiting examples of shingolipids are
N-acylsphingosine, N-Acylsphingomyelin, Forssman antigen.
[0247] Saccharolipids are compounds that contain both fatty acids
and sugars. The fatty acids are covalently bonded to a sugar
backbone. The sugar backbone can contain one or more sugars. The
fatty acids can bond to the sugars via either amide or ester bonds.
The sugar can be any sugar base. The fatty acid can be any fatty
acid as described elsewhere herein. The provided compositions can
comprise either natural or synthetic saccharolipids. Non-limiting
saccharolipids are UDP-3-O-(.beta.-hydroxymyristoyl)-GlcNAc, lipid
IV A, Kdo2-lipid A.
E. Linkers
[0248] Disclosed are linkers for associating components of the
disclosed compositions. Such linkers can be any molecule,
conjugate, composition, etc. that can be used to associate
components of the disclosed compositions. Generally, linkers can be
used to associate components other than surface molecules to
surface molecules. Useful linkers include materials that are
biocompatible, have low bioactivity, have low antigenicity, etc.
That is, such useful linker materials can serve the
linking/association function without adding unwanted bioreactivity
to the disclosed compositions. Many such materials are known and
used for similar linking and association functions. Polymer
materials are a particularly useful form of linker material. For
example, polyethylene glycols can be used.
[0249] Linkers are useful for achieving useful numbers and
densities of the components (such as homing molecules and membrane
perturbing molecules) on surface molecules. For example, linkers of
fibrous form are useful for increasing the number of components per
surface molecule or per a given area of the surface molecule.
Similarly, linkers having a branching form are useful for
increasing the number of components per surface molecule or per a
given area of the surface molecule. Linkers can also have a
branching fibrous form.
[0250] Linkers of different lengths can be used to bind the
disclosed components to surface molecules and to each other. A
flexible linker can function well even if relatively short, while a
stiffer linker may can be longer to allow effective exposure and
density. The length of a linker can refer to the number of atoms in
a continuous covalent chain between the attachment points on the
components being linked or to the length (in nanometers, for
example) of a continuous covalent chain between the attachment
points on the components being linked. Unless the context clearly
indicates otherwise, the length refers to the shortest continuous
covalent chain between the attachment points on the components
being linked not accounting for side chains, branches, or loops.
Due to flexibility of the linker, all of the linkers may not have
same distance from the surface molecule. Thus linkers with
different chain lengths can make the resulting composition more
effective (by increasing density, for example). Branched linkers
bearing multiple components also allow attachment of more than one
component at a given site of the surface molecule. Useful lengths
for linkers include at least, up to, about, exactly, or between 10,
15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150,
160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000,
7,000, 8,000, 9,000, and 10,000 atoms. Useful lengths for linkers
include at least, up to, about, exactly, or between 10, 15, 20, 25,
30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,
8,000, 9,000, and 10,000 nanometers. Any range of these lengths and
all lengths between the listed lengths are specifically
contemplated.
[0251] Hydrophilic or water-solubility linkers can increase the
mobility of the attached components. Examples of water-soluble,
biocompatible polymers which can serve as linkers include, but are
not limited to polymers such polyethylene glycol (PEG),
polyethylene oxide (PEO), polyvinyl alcohol, polyhydroxyethyl
methacrylate, polyacrylamide, and natural polymers such as
hyaluronic acid, chondroitin sulfate, carboxymethylcellulose, and
starch. Useful forms of branched tethers include star PEO and comb
PEO. Star PEO can be formed of many PEO "arms" emanating from a
common core.
[0252] Polyethylene glycols (PEGs) are simple, neutral polyethers
which have been given much attention in biotechnical and biomedical
applications (Milton Harris, J. (ed) "Poly(ethylene glycol)
chemistry, biotechnical and biomedical applications" Plenum Press,
New York, 1992). PEGs are soluble in most solvents, including
water, and are highly hydrated in aqueous environments, with two or
three water molecules bound to each ethylene glycol segment; this
hydration phenomenon has the effect of preventing adsorption either
of other polymers or of proteins onto PEG-modified surfaces.
Furthermore, PEGs may readily be modified and bound to other
molecules with only little effect on their chemistry. Their
advantageous solubility and biological properties are apparent from
the many possible uses of PEGs and copolymers thereof, including
block copolymers such as PEG-polyurethanes and PEG-polypropylenes.
Appropriate molecular weights for PEG linkers used in the disclosed
compositions can be from about 120 daltons to about 20 kilodaltons.
For example, PEGs can be at least, up to, about, exactly, or
between 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1000, 1200, 1400, 1500, 1600, 1800, 2000, 2500, 3000, 3500,
4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,
9500, 10,000, 20,000, 30,000, 40,000, and 50,000 daltons. Any range
of these masses and all masses between the listed masses are
specifically contemplated. PEGs are usually available as mixtures
of somewhat heterogeneous masses with a stated average mass
(PEG-5000, for example).
[0253] The disclosed compositions can be produced using any
suitable techniques. Many techniques, reactive groups, chemistries,
etc. for linking components of the types disclosed herein are known
and can be used with the disclosed components and compositions.
Examples of some techniques for producing the disclosed
compositions are described in the examples.
[0254] Protein crosslinkers that can be used to crosslink other
molecules, elements, moieties, etc. to the disclosed compositions,
surface molecules, homing molecules, membrane perturbing molecules,
internalization elements, tissue penetration elements, cargo
compositions, CendR elements, compositions, proteins, peptides,
amino acid sequences, etc. are known in the art and are defined
based on utility and structure and include DSS
(Disuccinimidylsuberate), DSP (Dithiobis(succinimidylpropionate)),
DTSSP (3,3'-Dithiobis (sulfosuccinimidylpropionate)), SULFO BSOCOES
(Bis[2-(sulfosuccinimdooxycarbonyloxy) ethyl]sulfone), BSOCOES
(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST
(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO
EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene
glycolbis(sulfosuccinimidylsuccinate)), DPDPB
(1,2-Di[3'-(2'-pyridyldithio) propionamido]butane), BSSS
(Bis(sulfosuccinimdyl) suberate), SMPB
(Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB
(Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS
(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS
(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB
(N-Succinimidyl(4-iodoacetyl) aminobenzoate), SULFO SIAB
(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC
(Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate),
SULFO SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate), NHS LC SPDP
(Succinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate),
SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio)
propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio)
propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate),
NHS IODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH
(4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH
(4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid hydrazide
hydrochloride), MBH (m-Maleimidobenzoic acid
hydrazidehydrochloride), SULFO EMCS
(N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide), EMCS
(N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI
(N-(p-Maleimidophenyl) isocyanate), KMUH
(N-(kappa-Maleimidoundecanoic acid) hydrazide), LC SMCC
(Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate-
)), SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide
ester), SMPH
(Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS
(N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMB S
(N-(gamma-Maleimidobutyrloxy) succinimide), DMP
(Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate
hydrochloride), MHBH (Wood's Reagent; Methyl-p-hydroxybenzimidate
hydrochloride, 98%), DMA (Dimethyladipimidate hydrochloride).
[0255] Components of the disclosed compositions, such as surface
molecules, homing molecules, membrane perturbing molecules,
internalization elements, tissue penetration elements, etc., can
also be coupled using, for example, maleimide coupling. By way of
illustration, components can be coupled to lipids by coupling to,
for example,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene
glycol).sub.2000; DSPE-PEG.sub.2000-maleimide] (Avanti Polar
Lipids) by making use of a free cysteine sulfhydryl group on the
component. The reaction can be performed, for example, in aqueous
solution at room temperature for 4 hours. This coupling chemistry
can be used to couple components of co-compositions and cargo
compositions.
[0256] Components of the disclosed compositions, such as surface
molecules, homing molecules, membrane perturbing molecules,
internalization elements, tissue penetration elements, etc., can
also be coupled using, for example, amino group-functionalized
dextran chemistry. Particles, such as, for example, nanoparticles,
nanoworms, and micelles, can be coated with amino group
functionalized dextran. Attachment of PEG to aminated particles
increases the circulation time, presumably by reducing the binding
of plasma proteins involved in opsonization (Moghimi et al., Pharm.
Rev. 53, 283-318 (2001)). The particles can have surface
modifications, for example, for reticuloendothelial system
avoidance (PEG) and homing (homing molecules), endosome escape
(pH-sensitive peptide; for example, Pirollo et al., Cancer Res. 67,
2938-43 (2007)), a detectable agent, a therapeutic compound, or a
combination. To accommodate all these functions on one particle,
optimization studies can be conducted to determine what proportion
of the available linking sites at the surface of the particles any
one of these elements should occupy to give the best combination of
targeting and payload delivery. The cell internalization and/or
tissue penetration of such compositions can be mediated by the
disclosed CendR elements, amino acid sequences, peptides, proteins,
molecules, conjugates, and compositions.
[0257] The provided peptides and polypeptides can have additional
N-terminal, C-terminal, or intermediate amino acid sequences, e.g.,
amino acid linkers or tags. The term "amino acid linker" refers to
an amino acid sequences or insertions that can be used to connect
or separate two distinct peptides, polypeptides, or polypeptide
fragments, where the linker does not otherwise contribute to the
essential function of the composition. The term "amino acid tag"
refers to a distinct amino acid sequence that can be used to detect
or purify the provided polypeptide, wherein the tag does not
otherwise contribute to the essential function of the composition.
The provided peptides and polypeptides can further have deleted
N-terminal, C-terminal or intermediate amino acids that do not
contribute to the essential activity of the peptides and
polypeptides.
[0258] Components can be directly or indirectly covalently bound to
surface molecules or each other by any functional group (e.g.,
amine, carbonyl, carboxyl, aldehyde, alcohol). For example, one or
more amine, alcohol or thiol groups on the components can be
reacted directly with isothiocyanate, acyl azide,
N-hydroxysuccinimide ester, aldehyde, epoxide, anhydride, lactone,
or other functional groups incorporated onto the surface molecules
or other components. Schiff bases formed between the amine groups
on the components and aldehyde groups on the surface molecule or
other components can be reduced with agents such as sodium
cyanoborohydride to form hydrolytically stable amine links
(Ferreira et al., J. Molecular Catalysis B: Enzymatic 2003, 21,
189-199). Components can be coupled to surface molecules and other
components by, for example, the use of a heterobifunctional silane
linker reagent, or by other reactions that activate functional
groups on either the surface molecule or the components.
[0259] Useful modes for linking components to surface molecules and
to other components include heterobifunctional linkers or spacers.
Such linkers can have both terminal amine and thiol reactive
functional groups for reacting amines on components with sulfhydryl
groups, thereby coupling the components in an oriented way. These
linkers can contain a variable number of atoms. Examples of such
linkers include, but are not limited to, N-Succinimidyl
3-(2-pyridyldithio)propionate (SPDP, 3- and 7-atom spacer),
long-chain-SPDP (12-atom spacer),
(Succinimidyloxycarbonyl-a-methyl-2-(2-pyridyldithio) toluene)
(SMPT, 8-atom spacer),
Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC,
11-atom spacer) and
Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
(sulfo-SMCC, 11-atom spacer), m-Maleimidobenzoyl-N
hydroxysuccinimide ester (MBS, 9-atom spacer),
N-(g-maleimidobutyryloxy)succinimide ester (GMBS, 8-atom spacer),
N-(g-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBS,
8-atom spacer), Succinimidyl 6-((iodoacetyl) amino) hexanoate
(SIAX, 9-atom spacer), Succinimidyl
6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)hexanoate (SIAXX, 16-atom
spacer), and p-nitrophenyl iodoacetate (NPIA, 2-atom spacer). One
ordinarily skilled in the art also will recognize that a number of
other coupling agents or links, with different number of atoms, may
be used.
[0260] Hydrophilic spacer atoms can be incorporated into linkers to
increase the distance between the reactive functional groups. For
example, polyethylene glycol (PEG) can be incorporated into
sulfo-GMBS. Hydrophilic molecules such as PEG have also been shown
to decrease non-specific binding (NSB) and increase hydrophilicity
of surfaces when covalently coupled. PEG can also be used as the
primary linker material.
[0261] Free amine groups of components can also be attached to
surface molecules or other components containing reactive amine
groups via homobifunctional linkers. Linkers such as
dithiobis(succinimidylpropionate) (DSP, 8-atom spacer),
disuccinimidyl suberate (DSS, 8-atom spacer), glutaraldehyde
(4-atom spacer), Bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone
(BSOCOES, 9-atom spacer), all requiring high pH, can be used for
this purpose. Examples of homobifunctional sulfhydryl-reactive
linkers include, but are not limited to,
1,4-Di-[3'-2'-pyridyldithio)propion-amido]butane (DPDPB, 16-atom
spacer) and Bismaleimidohexane (BMH, 14-atom spacer). For example,
these homobifunctional linkers are first reacted with a thiolated
surface in aqueous solution (for example PBS, pH 7.4), and then in
a second step, the thiolated antibody or protein is joined by the
link. Homo- and heteromultifunctional linkers can also be used.
[0262] Direct binding of components to thiol, amine, or carboxylic
acid functional groups on surface molecules and other components be
used to produce compositions which exhibit viral binding (due to
increased density of components, for example), resulting in
enhanced sensitivity.
[0263] As an example, when necessary to achieve high peptide
coupling density, additional amino groups can be added to the
surface molecules (such as commercially obtained SPIO) as follows:
First, to crosslink the particles before the amination step, 3 ml
of the colloid (.about.10 mg Fe/ml in double-distilled water) was
added to 5 ml of 5M NaOH and 2 ml of epichlorohydrin (Sigma, St.
Louis, Mo.). The mixture was agitated for 24 hours at room
temperature to promote interaction between the organic phase
(epichlorohydrin) and aqueous phase (dextran-coated particle
colloid). In order to remove excess epichlorohydrin, the reacted
mixture was dialyzed against double-distilled water for 24 hours
using a dialysis cassette (10,000 Da cutoff, Pierce, Rockford Ill.)
Amino groups were added to the surface of the particles as follows:
0.02 ml of concentrated ammonium hydroxide (30%) was added to 1 ml
of colloid (.about.10 mg Fe/ml). The mixture was agitated at room
temperature for 24 hours. The reacted mixture was dialyzed against
double-distilled water for 24 hours. To further rinse the
particles, the colloid was trapped on a MACS.RTM. Midi magnetic
separation column (Miltenyi Biotec, Auburn Calif.), rinsed with PBS
three times, and eluted from the column with 1 ml PBS.
[0264] To conjugate CGKRK peptide (and other peptides) to SPIO, the
particles were re-suspended at a concentration of 1 mg Fe/ml, and
heterobifunctional linker N-[a-maleimidoacetoxy]succinimide ester
(AMAS; Pierce) was added (2.5 mg linker per 2 mg Fe) under
vortexing. After incubation at room temperature for 40 min, the
particles were washed 3 times with 10 ml PBS on a MACS column The
peptide with free terminal cysteine was then added (100 .mu.g
peptide per 2 mg Fe). After incubation overnight at 4.degree. C.
the particles were washed again and re-suspended in PBS at a
concentration of 0.35 mg/ml of Fe). To quantify the number of
peptide molecules conjugated to the particles, a known amount of
stock or AMAS-activated particles was incubated with varying
amounts of the peptide. After completion of the incubation the
particles were pelleted at 100,000 G using Beckman TLA 100.3
ultracentrifuge rotor (30 min) and the amount of the unbound
peptide was quantified by fluorescence. To cleave the conjugated
peptide from the particles, the particles were incubated at
37.degree. C. overnight at pH 10. The concentration of free peptide
in the supernatant was determined by reading fluorescence and by
using the calibration curve obtained for the same peptide. The
fluorescence intensity of known amounts of particles was plotted as
a function of peptide conjugation density, and the slope equation
was used to determine conjugation density in different batches.
F. Peptides and Amino Acid Segments
[0265] In some forms, the homing molecule, cargo molecule,
internalization element, tissue penetration element, etc. can be or
include a peptide, peptidomimetic, and/or amino acid segment.
Unless the context indicates otherwise, reference herein to
"peptide" is intended to refer also to amino acid segments, which
can form a part of, or constitute an entire, peptide. The disclosed
peptides can be in isolated form. As used herein in reference to
the disclosed peptides, the term "isolated" means a peptide that is
in a form that is relatively free from material such as
contaminating polypeptides, lipids, nucleic acids and other
cellular material that normally is associated with the peptide in a
cell or that is associated with the peptide in a library or in a
crude preparation.
[0266] The disclosed peptides and amino acid segments can have any
suitable length. The disclosed peptides can have, for example, a
relatively short length of less than six, seven, eight, nine, ten,
12, 15, 20, 25, 30, 35 or 40 residues. The disclosed peptides also
can be useful in the context of a significantly longer sequence.
Thus, the peptides can have, for example, a length of up to 50,
100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In
particular embodiments, a peptide can have a length of at least 10,
20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further
embodiments, a peptide can have a length of 5 to 200 residues, 5 to
100 residues, 5 to 90 residues, 5 to 80 residues, 5 to 70 residues,
5 to 60 residues, 5 to 50 residues, 5 to 40 residues, 5 to 30
residues, 5 to 20 residues, 5 to 15 residues, 5 to 10 residues, 10
to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80
residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues,
10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 20 to 200
residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues,
20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40
residues or 20 to 30 residues. As used herein, the term "residue"
refers to an amino acid or amino acid analog.
[0267] The disclosed amino acid segments can have, for example, a
relatively short length of less than six, seven, eight, nine, ten,
12, 15, 20, 25, 30, 35 or 40 residues. The disclosed amino acid
segments also can be useful in the context of a significantly
longer sequence. Thus, the amino acid segments can have, for
example, a length of up to 50, 100, 150, 200, 250, 300, 400, 500,
1000 or 2000 residues. In particular embodiments, an amino acid
segment can have a length of at least 10, 20, 30, 40, 50, 60, 70,
80, 90, 100 or 200 residues. In further embodiments, an amino acid
segment can have a length of 5 to 200 residues, 5 to 100 residues,
5 to 90 residues, 5 to 80 residues, 5 to 70 residues, 5 to 60
residues, 5 to 50 residues, 5 to 40 residues, 5 to 30 residues, 5
to 20 residues, 5 to 15 residues, 5 to 10 residues, 10 to 200
residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues,
10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40
residues, 10 to 30 residues, 10 to 20 residues, 20 to 200 residues,
20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70
residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues
or 20 to 30 residues. As used herein, the term "residue" refers to
an amino acid or amino acid analog.
[0268] As this specification discusses various proteins, protein
sequences, peptides, peptides sequences, and amino acid sequences,
it is understood that the nucleic acids that can encode those
sequences are also disclosed. This would include all degenerate
sequences related to a specific protein sequence, i.e. all nucleic
acids having a sequence that encodes one particular protein
sequence as well as all nucleic acids, including degenerate nucleic
acids, encoding the disclosed variants and derivatives of the
protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence. The disclosed peptides and
proteins can be coupled to each other via peptide bonds to form
fusion peptides and proteins.
[0269] The disclosed peptides and amino acid segments can be
modified. As used herein, a "methylated derivative" of a protein,
peptide, amino acid segment, amino acid sequence, etc. refers to a
form of the protein, peptide, amino acid segment, amino acid
sequence, etc. that is methylated. Unless the context indicates
otherwise, reference to a methylated derivative of a protein,
peptide, amino acid segment, amino acid sequence, etc. does no
include any modification to the base protein, peptide, amino acid
segment, amino acid sequence, etc. other than methylation.
Methylated derivatives can also have other modifications, but such
modifications generally will be noted. For example, conservative
variants of an amino acid sequence would include conservative amino
acid substitutions of the based amino acid sequence. Thus,
reference to, for example, a "methylated derivative" of a specific
amino acid sequence "and conservative variants thereof" would
include methylated forms of the specific amino acid sequence and
methylated forms of the conservative variants of the specific amino
acid sequence, but not any other modifications of derivations. As
another example, reference to a methylated derivative of an amino
acid segment that includes amino acid substitutions would include
methylated forms of the amino acid sequence of the amino acid
segment and methylated forms of the amino acid sequence of the
amino acid segment include amino acid substitutions.
[0270] Protein variants and derivatives are well understood by
those of skill in the art and in can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues
Immunogenic fusion protein derivatives, such as those described in
the examples, are made by fusing a polypeptide sufficiently large
to confer immunogenicity to the target sequence by cross-linking in
vitro or by recombinant cell culture transformed with DNA encoding
the fusion. Deletions are characterized by the removal of one or
more amino acid residues from the protein sequence. Typically, no
more than about from 2 to 6 residues are deleted at any one site
within the protein molecule. These variants ordinarily are prepared
by site specific mutagenesis of nucleotides in the DNA encoding the
protein, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example M13 primer mutagenesis
and PCR mutagenesis Amino acid substitutions are typically of
single residues, but can occur at a number of different locations
at once; insertions usually will be on the order of about from 1 to
10 amino acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof can
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure.
[0271] As used herein in reference to a specified amino acid
sequence, a "conservative variant" is a sequence in which a first
amino acid is replaced by another amino acid or amino acid analog
having at least one biochemical property similar to that of the
first amino acid; similar properties include, for example, similar
size, charge, hydrophobicity or hydrogen-bonding capacity.
Conservative variants are also referred to herein as "conservative
amino acid substitutions," "conservative amino acid variants,"
"conservative substitutions," and similar phrase. A "conservative
derivative" of a reference sequence refers to an amino acid
sequence that differs from the reference sequences only in
conservative substitutions.
[0272] As an example, a conservative variant can be a sequence in
which a first uncharged polar amino acid is conservatively
substituted with a second (non-identical) uncharged polar amino
acid such as cysteine, serine, threonine, tyrosine, glycine,
glutamine or asparagine or an analog thereof. A conservative
variant also can be a sequence in which a first basic amino acid is
conservatively substituted with a second basic amino acid such as
arginine, lysine, histidine, 5-hydroxylysine, N-methyllysine or an
analog thereof. Similarly, a conservative variant can be a sequence
in which a first hydrophobic amino acid is conservatively
substituted with a second hydrophobic amino acid such as alanine,
valine, leucine, isoleucine, proline, methionine, phenylalanine or
tryptophan or an analog thereof. In the same way, a conservative
variant can be a sequence in which a first acidic amino acid is
conservatively substituted with a second acidic amino acid such as
aspartic acid or glutamic acid or an analog thereof; a sequence in
which an aromatic amino acid such as phenylalanine is
conservatively substituted with a second aromatic amino acid or
amino acid analog, for example, tyrosine; or a sequence in which a
first relatively small amino acid such as alanine is substituted
with a second relatively small amino acid or amino acid analog such
as glycine or valine or an analog thereof. For example, the
replacement of one amino acid residue with another that is
biologically and/or chemically similar is known to those skilled in
the art as a conservative substitution. For example, a conservative
substitution would be replacing one hydrophobic residue for
another, or one polar residue for another. The substitutions
include combinations such as, for example, Gly, Ala; Val, Ile, Leu;
Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such
conservatively substituted variations of each explicitly disclosed
sequence are included within the mosaic polypeptides provided
herein. It is understood that conservative variants of the
disclosed amino acid sequences can encompass sequences containing,
for example, one, two, three, four or more amino acid substitutions
relative to the reference sequence, and that such variants can
include naturally and non-naturally occurring amino acid
analogs.
[0273] Substitutional variants are those in which at least one
residue has been removed and a different residue inserted in its
place. Examples of such substitutions, referred to as conservative
substitutions, can generally be made in accordance with the
following Table 2.
TABLE-US-00002 TABLE 2 Amino Acid Substitutions Original Residue
Exemplary Conservative Substitutions, others are known in the art.
Ala Ser Arg Lys; Gln Asn Gln; His Asp Glu Cys Ser Gln Asn, Lys Glu
Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln Met
Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val
Ile; Leu
[0274] Substantial changes in function or immunological identity
can be made by selecting substitutions that are less conservative,
i.e., selecting residues that differ more significantly in their
effect on maintaining (a) the structure of the polypeptide backbone
in the area of the substitution, for example as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site or (c) the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in the protein properties will be those in which (a) a
hydrophilic residue, e.g. seryl or threonyl, is substituted for (or
by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,
valyl or alanyl; (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or
by) an electronegative residue, e.g., glutamyl or aspartyl; or (d)
a residue having a bulky side chain, e.g., phenylalanine, is
substituted for (or by) one not having a side chain, e.g., glycine,
in this case, (e) by increasing the number of sites for sulfation
and/or glycosylation. These can be referred to a less conservative
variants.
[0275] Peptides can have a variety of modifications. Modifications
can be used to change or improve the properties of the peptides.
For example, the disclosed peptides can be N-methylated,
O-methylated, S-methylated, C-methylated, or a combination at one
or more amino acids.
[0276] The amino and/or carboxy termini of the disclosed peptides
can be modified. Amino terminus modifications include methylation
(e.g., --NHCH.sub.3 or --N(CH.sub.3).sub.2), acetylation (e.g.,
with acetic acid or a halogenated derivative thereof such as
.alpha.-chloroacetic acid, .alpha.-bromoacetic acid, or
.alpha.-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group,
or blocking the amino terminus with any blocking group containing a
carboxylate functionality defined by RCOO-- or sulfonyl
functionality defined by R--SO.sub.2--, where R is selected from
the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and
the like, and similar groups. One can also incorporate a desamino
acid at the N-terminus (so that there is no N-terminal amino group)
to decrease susceptibility to proteases or to restrict the
conformation of the peptide compound. In preferred embodiments, the
N-terminus is acetylated with acetic acid or acetic anhydride.
[0277] Carboxy terminus modifications include replacing the free
acid with a carboxamide group or forming a cyclic lactam at the
carboxy terminus to introduce structural constraints. One can also
cyclize the disclosed peptides, or incorporate a desamino or
descarboxy residue at the termini of the peptide, so that there is
no terminal amino or carboxyl group, to decrease susceptibility to
proteases or to restrict the conformation of the peptide.
C-terminal functional groups of the disclosed peptides include
amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,
hydroxy, and carboxy, and the lower ester derivatives thereof, and
the pharmaceutically acceptable salts thereof.
[0278] One can replace the naturally occurring side chains of the
genetically encoded amino acids (or the stereoisomeric D amino
acids) with other side chains, for instance with groups such as
alkyl, lower (C.sub.1-6) alkyl, cyclic 4-, 5-, 6-, to 7-membered
alkyl, amide, amide lower alkyl amide di(lower alkyl), lower
alkoxy, hydroxy, carboxy and the lower ester derivatives thereof,
and with 4-, 5-, 6-, to 7-membered heterocyclic. In particular,
proline analogues in which the ring size of the proline residue is
changed from 5 members to 4, 6, or 7 members can be employed.
Cyclic groups can be saturated or unsaturated, and if unsaturated,
can be aromatic or non-aromatic. Heterocyclic groups preferably
contain one or more nitrogen, oxygen, and/or sulfur heteroatoms.
Examples of such groups include the furazanyl, furyl,
imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl,
morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.,
1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl,
pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,
pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl),
pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl,
thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These
heterocyclic groups can be substituted or unsubstituted. Where a
group is substituted, the substituent can be alkyl, alkoxy,
halogen, oxygen, or substituted or unsubstituted phenyl.
[0279] One can also readily modify peptides by phosphorylation, and
other methods [e.g., as described in Hruby, et al. (1990) Biochem
J. 268:249-262].
[0280] The disclosed peptides also serve as structural models for
non-peptidic compounds with similar biological activity. Those of
skill in the art recognize that a variety of techniques are
available for constructing compounds with the same or similar
desired biological activity as the lead peptide compound, but with
more favorable activity than the lead with respect to solubility,
stability, and susceptibility to hydrolysis and proteolysis [See,
Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243-252]. These
techniques include, but are not limited to, replacing the peptide
backbone with a backbone composed of phosphonates, amidates,
carbamates, sulfonamides, secondary amines, and N-methylamino
acids.
[0281] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include
CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH--
(cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and
--CHH.sub.2SO-- (These and others can be found in Spatola, A. F. in
Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,
B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);
Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide
Backbone Modifications (general review); Morley, Trends Pharm Sci
(1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res
14:177-185 (1979) (--CH.sub.2NH--, CH.sub.2CH.sub.2); Spatola et
al. Life Sci 38:1243-1249 (1986) (--CH H.sub.2--S); Hann J. Chem.
Soc Perkin Trans. I 307-314 (1982) (--CH--CH--, cis and trans);
Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH.sub.2--);
Jennings-White et al. Tetrahedron Lett 23:2533 (1982)
(--COCH.sub.2--); Szelke et al. European Appln, EP 45665 CA (1982):
97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay et al. Tetrahedron.
Lett 24:4401-4404 (1983) (--C(OH)CH.sub.2--); and Hruby Life Sci
31:189-199 (1982) (--CH.sub.2--S--); each of which is incorporated
herein by reference. A particularly preferred non-peptide linkage
is --CH.sub.2NH--. It is understood that peptide analogs can have
more than one atom between the bond atoms, such as .beta.-alanine,
.gamma.-aminobutyric acid, and the like.
[0282] Substitutional or deletional mutagenesis can be employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation
(Ser or Thr). Deletions of cysteine or other labile residues also
can be desirable. Deletions or substitutions of potential
proteolysis sites, e.g. Arg, can be accomplished, for example, by
deleting one of the basic residues or substituting one by
glutaminyl or histidyl residues.
[0283] Certain post-translational derivatizations can be the result
of the action of recombinant host cells on the expressed
polypeptide. Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the o-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco pp
79-86 [1983]), acetylation of the N-terminal amine and, in some
instances, amidation of the C-terminal carboxyl.
[0284] It is understood that one way to define the variants and
derivatives of the disclosed amino acids sequences, amino acid
segments, peptides, proteins, etc. herein is through defining the
variants and derivatives in terms of homology/identity to specific
known sequences. For example, specifically disclosed are variants
of these and other amino acids sequences, amino acid segments,
peptides, proteins, etc. herein disclosed which have at least, 70%
or 75% or 80% or 85% or 90% or 95% homology to the stated sequence.
Those of skill in the art readily understand how to determine the
homology of two proteins. For example, the homology can be
calculated after aligning the two sequences so that the homology is
at its highest level.
[0285] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
can be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0286] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment.
[0287] It is understood that the description of conservative
variants and homology can be combined together in any combination,
such as embodiments that have at least 70% homology to a particular
sequence wherein the variants are conservative variants.
[0288] As this specification discusses various amino acids
sequences, amino acid segment sequences, peptide sequences, protein
sequences, etc., it is understood that nucleic acids that can
encode those sequences are also disclosed. This would include all
degenerate sequences related to a specific amino acid sequence,
i.e. all nucleic acids having a sequence that encodes one
particular amino acid sequence as well as all nucleic acids,
including degenerate nucleic acids, encoding the disclosed variants
and derivatives of the amino acid sequences. Thus, while each
particular nucleic acid sequence may not be written out herein, it
is understood that each and every sequence is in fact disclosed and
described herein through the disclosed amino acid sequences.
[0289] Also disclosed are bifunctional peptides, which contain the
homing peptide fused to a second peptide having a separate
function. Such bifunctional peptides have at least two functions
conferred by different portions of the full-length molecule and
can, for example, display anti-angiogenic activity or pro-apoptotic
activity in addition to the ability to home to a target.
[0290] Also disclosed are isolated multivalent peptides that
include at least two subsequences each independently containing a
peptide or amino acid segment. The multivalent peptide can have,
for example, at least three, at least five or at least ten of such
subsequences each independently containing a peptide. In particular
embodiments, the multivalent peptide can have two, three, four,
five, six, seven, eight, nine, ten, fifteen or twenty identical or
non-identical subsequences. This is in addition to the multiple
homing molecules and, for example, multiple membrane disrupting
molecules that can comprise the disclosed compositions. In a
further embodiment, the multivalent peptide can contain identical
subsequences, such as repeats of a specified amino acid sequence.
In a further embodiment, the multivalent peptide contains
contiguous identical or non-identical subsequences, which are not
separated by any intervening amino acids.
[0291] As used herein, the term "peptide" is used broadly to mean
peptides, proteins, fragments of proteins and the like. The term
"peptidomimetic," as used herein, means a peptide-like molecule
that has the activity of the peptide upon which it is structurally
based. Such peptidomimetics include chemically modified peptides,
peptide-like molecules containing non-naturally occurring amino
acids, and peptoids and have an activity such as selective
interaction with a target of the peptide upon which the
peptidomimetic is derived (see, for example, Goodman and Ro,
Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry
and Drug Discovery" Vol. 1 (ed. M. E. Wolff; John Wiley & Sons
1995), pages 803-861).
[0292] A variety of peptidomimetics are known in the art including,
for example, peptide-like molecules which contain a constrained
amino acid, a non-peptide component that mimics peptide secondary
structure, or an amide bond isostere. A peptidomimetic that
contains a constrained, non-naturally occurring amino acid can
include, for example, an .alpha.-methylated amino acid;
.alpha.,.alpha..-dialkylglycine or .alpha.-aminocycloalkane
carboxylic acid; an N.sup..alpha.--C.sup..alpha. cyclized amino
acid; an N.sup..alpha..-methylated amino acid; a .beta.- or
.gamma.-amino cycloalkane carboxylic acid; an
.alpha.,.beta.-unsaturated amino acid; a .beta.,.beta.-dimethyl or
.beta.-methyl amino acid; a .beta.-substituted-2,3-methano amino
acid; an N--C.sup..epsilon. or C.sup..alpha.--C.sup..DELTA.
cyclized amino acid; a substituted proline or another amino acid
mimetic. A peptidomimetic which mimics peptide secondary structure
can contain, for example, a non-peptidic .beta.-turn mimic;
.gamma.-turn mimic; mimic of .beta.-sheet structure; or mimic of
helical structure, each of which is well known in the art. A
peptidomimetic also can be a peptide-like molecule which contains,
for example, an amide bond isostere such as a retro-inverso
modification; reduced amide bond; methylenethioether or
methylene-sulfoxide bond; methylene ether bond; ethylene bond;
thioamide bond; trans-olefin or fluoroolefin bond;
1,5-disubstituted tetrazole ring; ketomethylene or
fluoroketomethylene bond or another amide isostere. One skilled in
the art understands that these and other peptidomimetics are
encompassed within the meaning of the term "peptidomimetic" as used
herein.
[0293] Methods for identifying a peptidomimetic are well known in
the art and include, for example, the screening of databases that
contain libraries of potential peptidomimetics. As an example, the
Cambridge Structural Database contains a collection of greater than
300,000 compounds that have known crystal structures (Allen et al.,
Acta Crystalloqr. Section B, 35:2331 (1979)). This structural
depository is continually updated as new crystal structures are
determined and can be screened for compounds having suitable
shapes, for example, the same shape as a disclosed peptide, as well
as potential geometrical and chemical complementarity to a target
molecule. Where no crystal structure of a peptide or a target
molecule that binds the peptide is available, a structure can be
generated using, for example, the program CONCORD (Rusinko et al.,
J. Chem. Inf. Comput. Sci. 29:251 (1989)). Another database, the
Available Chemicals Directory (Molecular Design Limited,
Information Systems; San Leandro Calif.), contains about 100,000
compounds that are commercially available and also can be searched
to identify potential peptidomimetics of a peptide, for example,
with activity in selectively interacting with cancerous cells.
G. Pharmaceutical Compositions and Carriers
[0294] The disclosed compositions can be administered in vivo
either alone or in a pharmaceutically acceptable carrier. By
"pharmaceutically acceptable" is meant a material that is not
biologically or otherwise undesirable, i.e., the material can be
administered to a subject, along with the composition disclosed
herein, without causing any undesirable biological effects or
interacting in a deleterious manner with any of the other
components of the pharmaceutical composition in which it is
contained. The carrier would naturally be selected to minimize any
degradation of the active ingredient and to minimize any adverse
side effects in the subject, as would be well known to one of skill
in the art. The materials can be in solution, suspension (for
example, incorporated into microparticles, liposomes, or
cells).
[0295] 1. Pharmaceutically Acceptable Carriers
[0296] The compositions disclosed herein can be used
therapeutically in combination with a pharmaceutically acceptable
carrier.
[0297] Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.
R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically,
an appropriate amount of a pharmaceutically-acceptable salt is used
in the formulation to render the formulation isotonic. Examples of
the pharmaceutically-acceptable carrier include, but are not
limited to, saline, Ringer's solution and dextrose solution. The pH
of the solution is preferably from about 5 to about 8, and more
preferably from about 7 to about 7.5. Further carriers include
sustained release preparations such as semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, liposomes or
microparticles. It will be apparent to those persons skilled in the
art that certain carriers can be more preferable depending upon,
for instance, the route of administration and concentration of
composition being administered.
[0298] Pharmaceutical carriers are known to those skilled in the
art. These most typically would be standard carriers for
administration of drugs to humans, including solutions such as
sterile water, saline, and buffered solutions at physiological pH.
The compositions can be administered intramuscularly or
subcutaneously. Other compounds will be administered according to
standard procedures used by those skilled in the art.
[0299] Pharmaceutical compositions can include carriers,
thickeners, diluents, buffers, preservatives, surface active agents
and the like in addition to the molecule of choice. Pharmaceutical
compositions can also include one or more active ingredients such
as antimicrobial agents, antiinflammatory agents, anesthetics, and
the like.
[0300] The pharmaceutical composition can be administered in a
number of ways depending on whether local or systemic treatment is
desired, and on the area to be treated. Administration can be
topically (including ophthalmically, vaginally, rectally,
intranasally), orally, by inhalation, or parenterally, for example
by intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection. The disclosed antibodies can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, or transdermally.
[0301] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives can also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0302] Formulations for topical administration can include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0303] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0304] Some of the compositions can be administered as a
pharmaceutically acceptable acid- or base-addition salt, formed by
reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
H. Compositions with Similar Functions
[0305] It is understood that the compositions disclosed herein have
certain functions, such as binding to clots or enhancing clot
formation. Disclosed herein are certain structural requirements for
performing the disclosed functions, and it is understood that there
are a variety of structures which can perform the same function
which are related to the disclosed structures, and that these
structures will ultimately achieve the same result, for example
stimulation or inhibition.
I. Kits
[0306] Disclosed herein are kits that are drawn to reagents that
can be used in practicing the methods disclosed herein. The kits
can include any reagent or combination of reagent discussed herein
or that would be understood to be required or beneficial in the
practice of the disclosed methods. For example, the kits can
include the compositions disclosed herein.
J. Mixtures
[0307] Whenever the method involves mixing or bringing into contact
compositions or components or reagents, performing the method
creates a number of different mixtures. For example, if the method
includes 3 mixing steps, after each one of these steps a unique
mixture is formed if the steps are performed separately. In
addition, a mixture is formed at the completion of all of the steps
regardless of how the steps were performed. The present disclosure
contemplates these mixtures, obtained by the performance of the
disclosed methods as well as mixtures containing any disclosed
reagent, composition, or component, for example, disclosed
herein.
K. Systems
[0308] Disclosed are systems useful for performing, or aiding in
the performance of, the disclosed method. Systems generally
comprise combinations of articles of manufacture such as
structures, machines, devices, and the like, and compositions,
compounds, materials, and the like. Such combinations that are
disclosed or that are apparent from the disclosure are
contemplated.
L. Peptide Synthesis
[0309] The compositions disclosed herein and the compositions
necessary to perform the disclosed methods can be made using any
method known to those of skill in the art for that particular
reagent or compound unless otherwise specifically noted.
[0310] One method of producing the disclosed proteins is to link
two or more peptides or polypeptides together by protein chemistry
techniques. For example, peptides or polypeptides can be chemically
synthesized using currently available laboratory equipment using
either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc
(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,
Foster City, Calif.). One skilled in the art can readily appreciate
that a peptide or polypeptide corresponding to the disclosed
proteins, for example, can be synthesized by standard chemical
reactions. For example, a peptide or polypeptide can be synthesized
and not cleaved from its synthesis resin whereas the other fragment
of a peptide or protein can be synthesized and subsequently cleaved
from the resin, thereby exposing a terminal group which is
functionally blocked on the other fragment. By peptide condensation
reactions, these two fragments can be covalently joined via a
peptide bond at their carboxyl and amino termini, respectively, to
form an antibody, or fragment thereof. (Grant G A (1992) Synthetic
Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky
M and Trost B., Ed. (1993) Principles of Peptide Synthesis.
Springer-Verlag Inc., NY (which is herein incorporated by reference
at least for material related to peptide synthesis). Alternatively,
the peptide or polypeptide is independently synthesized in vivo as
described herein. Once isolated, these independent peptides or
polypeptides can be linked to form a peptide or fragment thereof
via similar peptide condensation reactions.
[0311] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide-thioester with another unprotected peptide
segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site (Baggiolini M et al.
(1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J. Biol. Chem.,
269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128
(1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
[0312] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
METHODS
[0313] Disclosed are methods useful for delivering significant
amounts of compounds o interest to targeted cells and tissues. The
disclosed methods are useful, for example, to deliver to targeted
cells and tissues an effective amount of compounds that are
excessively toxic. Also disclosed are methods comprising, for
example, administering to a subject the disclosed compositions.
Also disclosed are methods of detecting, measuring, imaging, etc.
cells and tissues comprising, for example, administering to a
subject the disclosed compositions and detecting, measuring,
imaging, etc. the composition.
[0314] The homing molecules can home to targets of interest, such
as cells and tissues of interest. For example, the homing molecules
can home to tumor vasculature. The homing molecules can selectively
home to targets of interest, such as cells and tissues of interest.
For example, the homing molecules can selectively homes to tumor
vasculature. The composition can home to one or more of the sites
to be targeted. The composition can be internalized in cells. The
composition can penetrate tissue. The composition can be
internalized into cells at the targeted site. The composition can
be penetrate tissue at the targeted site. The composition can, for
example be internalized into cancer cells. The composition can, for
example, penetrate tumor tissue. The composition can, for example,
bind inside tumor blood vessels.
[0315] In some forms, the composition can have a therapeutic
effect. In some forms, the composition can reduce tumor growth. In
some forms, the therapeutic effect can be a slowing in the increase
of or a reduction of tumor burden. In some forms, the therapeutic
effect can be a slowing of the increase of or reduction of tumor
size. In some forms, the subject can have one or more sites
targeted, wherein the composition can home to one or more of the
sites targeted. In some forms, the subject can have a tumor,
wherein the composition can have a therapeutic effect on the
tumor.
[0316] In some forms, the composition can further comprise one or
more internalization elements. In some forms, one or more of the
homing molecules can comprise one or more of the internalization
elements. In some forms, one or more of the membrane perturbing
molecules can comprise one or more of the internalization elements.
In some forms, the surface molecule can comprise one or more of the
internalization elements not comprised in either the homing
molecules or the membrane perturbing molecules. In some forms, the
composition can further comprise one or more tissue penetration
elements. In some forms, one or more of the tissue penetration
elements can be comprised in an internalization element. In some
forms, the tissue penetration element can be a CendR element.
[0317] In some forms, the composition can further comprise one or
more moieties. In some forms, the moieties can be independently
selected from the group consisting of an anti-angiogenic agent, a
pro-angiogenic agent, a cancer chemotherapeutic agent, a cytotoxic
agent, an anti-inflammatory agent, an anti-arthritic agent, a
polypeptide, a nucleic acid molecule, a small molecule, an image
contrast agent, a fluorophore, fluorescein, rhodamine, a
radionuclide, indium-111, technetium-99, carbon-11, and carbon-13.
In some forms, at least one of the moieties can be a therapeutic
agent. In some forms, the therapeutic agent can be iRGD, RGD,
Abraxane, paclitaxel, taxol, or a combination. In some forms, at
least one of the moieties can be a detectable agent. In some forms,
the detectable agent can be FAM.
[0318] In some forms, the composition can have a therapeutic
effect. This can be achieved by the delivery of therapeutic cargo
molecules to the target site. The therapeutic effect can be a
slowing in the increase of or a reduction of tumor burden. This
slowing in the increase of, or reduction in the tumor burden, can
be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%,
500%, 600%, 700%, 800%, 900%, or 1000% or more improvement in the
increase of, or reduction in the tumor burden of, compared with a
non-treated tumor, or a tumor treated by a different method.
[0319] The disclosed compositions can be used to treat any disease
where uncontrolled cellular proliferation occurs such as cancers. A
non-limiting list of different types of cancers can be as follows:
lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas,
carcinomas of solid tissues, squamous cell carcinomas,
adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas,
neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas,
hypoxic tumors, myelomas, AIDS-related lymphomas or sarcomas,
metastatic cancers, or cancers in general.
[0320] A representative but non-limiting list of cancers that the
disclosed compositions can be used to treat is the following:
lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides,
Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer,
nervous system cancer, head and neck cancer, squamous cell
carcinoma of head and neck, kidney cancer, lung cancers such as
small cell lung cancer and non-small cell lung cancer,
neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,
prostate cancer, skin cancer, liver cancer, melanoma, squamous cell
carcinomas of the mouth, throat, larynx, and lung, colon cancer,
cervical cancer, cervical carcinoma, breast cancer, and epithelial
cancer, renal cancer, genitourinary cancer, pulmonary cancer,
esophageal carcinoma, head and neck carcinoma, large bowel cancer,
hematopoietic cancers; testicular cancer; colon and rectal cancers,
prostatic cancer, or pancreatic cancer.
[0321] The disclosed compositions can also be administered
following decoy particle pretreatment to reduce uptake of the
compositions by reticuloendothelial system (RES) tissues. Such
decoy particle pretreatment can prolong the blood half-life of the
particles and increases tumor targeting.
[0322] The method can further comprise, following administering,
detecting the disclosed compositions. The disclosed compositions
can be detected by fluorescence, CT scan, PET or MRI. The disclosed
compositions can be detected by fluorescence. The disclosed
compositions can conjugate with tumor vasculature or a tumor in a
subject.
[0323] By "treatment" is meant the medical management of a patient
with the intent to cure, ameliorate, stabilize, or prevent a
disease, pathological condition, or disorder. This term includes
active treatment, that is, treatment directed specifically toward
the improvement of a disease, pathological condition, or disorder,
and also includes causal treatment, that is, treatment directed
toward removal of the cause of the associated disease, pathological
condition, or disorder. In addition, this term includes palliative
treatment, that is, treatment designed for the relief of symptoms
rather than the curing of the disease, pathological condition, or
disorder; preventative treatment, that is, treatment directed to
minimizing or partially or completely inhibiting the development of
the associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0324] As used herein, "subject" includes, but is not limited to,
animals, plants, bacteria, viruses, parasites and any other
organism or entity that has nucleic acid. The subject may be a
vertebrate, more specifically a mammal (e.g., a human, horse, pig,
rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig
or rodent), a fish, a bird or a reptile or an amphibian. In
particular, pets and livestock can be a subject. The subject can be
an invertebrate, such as a worm or an arthropod (e.g., insects and
crustaceans). The term does not denote a particular age or sex.
Thus, adult and newborn subjects, as well as fetuses, whether male
or female, are intended to be covered. A patient refers to a
subject afflicted with a disease or disorder. The term "patient"
includes human and veterinary subjects. In the context of
endometriosis and endometriosis cells, it is understood that a
subject is a subject that has or can have endometriosis and/or
endometriosis cells.
[0325] In one aspect, the compounds described herein can be
administered to a subject comprising a human or an animal
including, but not limited to, a mouse, dog, cat, horse, bovine or
ovine and the like, that is in need of alleviation or amelioration
from a recognized medical condition.
[0326] By the term "effective amount" of a compound as provided
herein is meant a nontoxic but sufficient amount of the compound to
provide the desired result. As will be pointed out below, the exact
amount required will vary from subject to subject, depending on the
species, age, and general condition of the subject, the severity of
the disease that is being treated, the particular compound used,
its mode of administration, and the like. Thus, it is not possible
to specify an exact "effective amount." However, an appropriate
effective amount can be determined by one of ordinary skill in the
art using only routine experimentation.
[0327] The dosages or amounts of the compounds described herein are
large enough to produce the desired effect in the method by which
delivery occurs. The dosage should not be so large as to cause
adverse side effects, such as unwanted cross-reactions,
anaphylactic reactions, and the like. Generally, the dosage will
vary with the age, condition, sex and extent of the disease in the
subject and can be determined by one of skill in the art. The
dosage can be adjusted by the individual physician based on the
clinical condition of the subject involved. The dose, schedule of
doses and route of administration can be varied.
[0328] The efficacy of administration of a particular dose of the
compounds or compositions according to the methods described herein
can be determined by evaluating the particular aspects of the
medical history, signs, symptoms, and objective laboratory tests
that are known to be useful in evaluating the status of a subject
in need for the treatment of cancer or other diseases and/or
conditions. These signs, symptoms, and objective laboratory tests
will vary, depending upon the particular disease or condition being
treated or prevented, as will be known to any clinician who treats
such patients or a researcher conducting experimentation in this
field. For example, if, based on a comparison with an appropriate
control group and/or knowledge of the normal progression of the
disease in the general population or the particular individual: (1)
a subject's physical condition is shown to be improved (e.g., a
tumor has partially or fully regressed), (2) the progression of the
disease or condition is shown to be stabilized, or slowed, or
reversed, or (3) the need for other medications for treating the
disease or condition is lessened or obviated, then a particular
treatment regimen will be considered efficacious.
[0329] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material can
be administered to an individual along with the selected compound
without causing any undesirable biological effects or interacting
in a deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained.
[0330] Any of the compounds having the formula I can be used
therapeutically in combination with a pharmaceutically acceptable
carrier. The compounds described herein can be conveniently
formulated into pharmaceutical compositions composed of one or more
of the compounds in association with a pharmaceutically acceptable
carrier. See, e.g., Remington's Pharmaceutical Sciences, latest
edition, by E.W. Martin Mack Pub. Co., Easton, Pa., which discloses
typical carriers and conventional methods of preparing
pharmaceutical compositions that can be used in conjunction with
the preparation of formulations of the compounds described herein
and which is incorporated by reference herein. These most typically
would be standard carriers for administration of compositions to
humans. In one aspect, humans and non-humans, including solutions
such as sterile water, saline, and buffered solutions at
physiological pH. Other compounds will be administered according to
standard procedures used by those skilled in the art.
[0331] The pharmaceutical compositions described herein can
include, but are not limited to, carriers, thickeners, diluents,
buffers, preservatives, surface active agents and the like in
addition to the molecule of choice. Pharmaceutical compositions can
also include one or more active ingredients such as antimicrobial
agents, antiinflammatory agents, anesthetics, and the like.
[0332] The compounds and pharmaceutical compositions described
herein can be administered to the subject in a number of ways
depending on whether local or systemic treatment is desired, and on
the area to be treated. Thus, for example, a compound or
pharmaceutical composition described herein can be administered as
an ophthalmic solution and/or ointment to the surface of the eye.
Moreover, a compound or pharmaceutical composition can be
administered to a subject vaginally, rectally, intranasally,
orally, by inhalation, or parenterally, for example, by
intradermal, subcutaneous, intramuscular, intraperitoneal,
intrarectal, intraarterial, intralymphatic, intravenous,
intrathecal and intratracheal routes. Parenteral administration, if
used, is generally characterized by injection. Injectables can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution or suspension in
liquid prior to injection, or as emulsions. A more recently revised
approach for parenteral administration involves use of a slow
release or sustained release system such that a constant dosage is
maintained. See, e.g., U.S. Pat. No. 3,610,795, which is
incorporated by reference herein.
[0333] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions which
can also contain buffers, diluents and other suitable additives.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives can also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0334] Formulations for topical administration can include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like can be necessary or
desirable.
[0335] Compositions for oral administration can include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders can be desirable.
EXAMPLES
[0336] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
disclosure. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
A. Example 1
Nanoparticle Homing to Tumors
[0337] Glioblastomas multiforme (GBM) are the most common and
lethal form of intracranial tumors. They account for approximately
70% of the 22,500 new cases of malignant primary brain tumors that
are diagnosed in adults in the United States each year. Although
relatively uncommon, malignant gliomas are associated with
disproportionately high morbidity and mortality (median survival is
only 12 to 15 months). Malignant gliomas are among the most
vascular of human tumors. Tumor vasculature has proven to be
particularly well suited as a site for receptor-based targeting. It
expresses a multitude of molecules that are not expressed in the
vessels of normal tissues. A peptide, CGKRK (Hoffman, J. A., et al.
Progressive vascular changes in a transgenic mouse model of
squamous cell carcinoma. Cancer Cell 4, 383-391 (2003)), binds to
the blood vessels in various kinds of tumors. Experiments showed
that intravenously injected CGKRK peptide effectively homes to
lentiviral (H-RasV12-sip53)-induced glioma in mice. The CGKRK
peptide was coupled to the alpha-helical amphipathic peptide
.sub.D[KLAKLAK].sub.2, which is toxic to eukaryotic cells if it
internalized into the cells (Ellerby, H. M., et al. Anti-cancer
activity of targeted pro-apoptotic peptides. Nature Medicine 5,
1032-1038 (1999)). The chimeric peptide, when added to actively
growing human umbilical vein endothelial cells (HUVEC) or U87
glioma cells colocalized with mitochondria, whereas
.sub.D[KLAKLAK].sub.2 did not. Iron oxide nanoworms (NW) coated
with the chimeric .sub.D[KLAKLAK].sub.2-CGKRK peptide were
100-200-fold more toxic to the HUVEC and U87 cells than the free
peptide in vitro and specifically accumulated in the blood vessels
of glioma. Treatment of lentiviral induced glioma in mice with the
.sub.D[KLAKLAK].sub.2-CGKRK-PEG-NW inhibited tumor growth and
showed a significant survival increase compared to control-treated
mice.
[0338] Mice bearing brain tumors (RAS-sip53 induced brain tumors
into the right hippocampus) were intravenously injected with 200
microg of FAM-labeled CGKRK peptides and allowed to circulate for 3
hours. The mice were perfused through the heart with PBS, and the
organs were collected. CGKRK peptide accumulated in glioblastoma
compared to normal brain tissue. The dotted line outlines the
tumor. Magnification .times.200.
[0339] Cultured cells were treated CGKRK, .sub.D(KLAKLAK).sub.2, or
.sub.D(KLAKLAK).sub.2CGKRK peptide. The cells were incubated with
peptide for 24 hrs and cell death was quantified by MTT assays
(n=3). FIGS. 2A, 2B and 2C show cytotoxicity of
.sub.D(KLAKLAK).sub.2CGKRK peptide in cell lines. FIGS. 2A and 2B
show the cytotoxicity of .sub.D(KLAKLAK).sub.2CGKRK peptide in
HUVEC (A) and T3 (B) cells. Statistical analyses were performed
with Student's t-test. Error bars, s.e.m.
[0340] Confocal microscopic images of HUVEC cells incubated for 2 h
at 37.degree. C. with KAKEC-NW (SEQ ID NO:135), or CGKRK-NW, or
.sub.D(KLAKLAK).sub.2-NW, or .sub.D(KLAKLAK).sub.2CGKRK-NW were
prepared. Subcellular localization of nanoworms was identified in
HUVEC cells. There is a high sub-colocalization of CGKRK-NW and
.sub.D(KLAKLAK).sub.2CGKRK-NW with mitochondria marker.
[0341] Confocal microscopic images of HUVEC cells incubated for 2 h
at 37.degree. C. with CGKRK-NW or .sub.D(KLAKLAK).sub.2CGKRK-NW
were prepared. Competition of subcellular localization of nanoworms
in HUVEC cells was seen with both CGKRK-NW and
.sub.D(KLAKLAK).sub.2CGKRK-NW treated cells. 10.times. non-labeled
peptide-NW was added to the cells 15 min before adding the labeled
peptide-NW. Cultured HUVEC cells were treated with non-targeted
.sub.D(KLAKLAK).sub.2 conjugated NW (.sub.D(KLAKLAK).sub.2-NW),
CREKA conjugated NW (CREKA-NW), CGKRK conjugated NW (CGKRK-NW), or
CGKRK-.sub.D(KLAKLAK).sub.2 conjugates NW
(.sub.D(KLAKLAK).sub.2-CGKRK-NW). FIGS. 3A and 3B show cytotoxicity
of .sub.D(KLAKLAK).sub.2CGKRK conjugated with NW in HUVEC cells.
The cells were incubated with NW for 48 hrs without washing (A) or
the NW were washed after 20 min (B) and cell death was quantified
by MTT assays (n=3). Statistical analyses were performed with
Student's t-test. Error bars, s.e.m.
[0342] Cultured T3 cells were treated with non-targeted
.sub.D(KLAKLAK).sub.2 conjugated NW (.sub.D(KLAKLAK).sub.2-NW),
CREKA conjugated NW (CREKA-NW), CGKRK conjugated NW (CGKRK-NW), or
.sub.D(KLAKLAK).sub.2CGKRK conjugated with NW
(.sub.D(KLAKLAK).sub.2CGKRK-NW). The cells were incubated with NW
for 48 hrs and cell death was quantified by MTT assay. FIG. 4 shows
cytotoxicity of .sub.D(KLAKLAK).sub.2CGKRK conjugated with NW in T3
cells.
[0343] Confocal microscopic images of U87 cells incubated for 2 h
at 37.degree. C. with CGKRK-NW, or .sub.D(KLAKLAK).sub.2-NW, or
CGKRK-.sub.D(KLAKLAK).sub.2-NW (Chimera-NW) were prepared.
Subcellular localization of nanoworms was identified in U87 cells.
There is high sub-colocalization of CGKRK-NW and
.sub.D(KLAKLAK).sub.2CGKRK-NW with mitochondria.
[0344] Cultured U87 cells were treated with non-targeted
.sub.D(KLAKLAK).sub.2 conjugated NW, KAKEC (SEQ ID NO:135)
conjugated NW (KAKEC-NW), CGKRK conjugated NW (CGKRK-NW), or
CGKRK-.sub.D(KLAKLAK).sub.2 conjugated with NW
(.sub.D(KLAKLAK).sub.2CGKRK-NW). The cells were incubated with NW
for 24 or 48 hrs and cell death was quantified by MTT assays. FIG.
5 shows cytotoxicity of .sub.D(KLAKLAK).sub.2CGKRK conjugated with
NW in U87 cells. These results are almost the same results seen
with U251 which had 50-60% cell viability.
[0345] NW coated with .sub.D(KLAKLAK).sub.2CGKRK via a 5-kDa
PEG-linker were cleaved from the particles using DTT and the amount
of peptide present on the particle was calculated to compare the
amount of free peptide versus the peptide coated nanoparticle IC50
values. FIG. 6 shows the IC50 of .sub.D(KLAKLAK).sub.2CGKRK peptide
versus peptide on nanoworms.
[0346] HUVEC cells were left untreated (Control) or treated for 24,
48 and 72 hrs with an irrelevant peptide-NW (CREKA-NW) or the
.sub.D(KLAKLAK).sub.2CGKRK-NW. Cells were incubated with Annexin
V-PE in a buffer containing 7-Amino-actinomycin (7-AAD) and
analyzed by flow cytometry. FIG. 7 shows .sub.D(KLAKLAK).sub.2CGKRK
conjugated with NW induced apoptosis in HUVEC cells. The percentage
of Annexin V positive cells (apoptotic cells plus end stage
apoptosis or already dead cells) is indicated in each graph.
[0347] T3 cells (tumor endothelial cells) were left untreated
(Control) or treated for 24 and 48 hrs with an irrelevant
peptide-NW (CREKA-NW) or the .sub.D(KLAKLAK).sub.2CGKRK-NW. Cells
were incubated with Annexin V-PE in a buffer containing
7-Amino-actinomycin (7-AAD) and analyzed by flow cytometry. FIG. 8
shows .sub.D(KLAKLAK).sub.2CGKRK conjugated with NW induced
apoptosis in T3 cells. The percentage of Annexin V positive cells
(apoptotic cells plus end stage apoptosis or already dead cells) is
indicated in each graph.
[0348] Primary HUVECs were plated on growth factor reduced matrigel
in 5% FCS medium alone (control), or containing CGKRK-NW (10
microg/ml), or containing .sub.D(KLAKLAK).sub.2CGKRK-NW (5 and 10
microg/ml). The formation of networks of capillary-like structures
was viewed by phase contrast-microscopy at 40.times. magnification
24 h after plating. FIG. 9 shows .sub.D(KLAKLAK).sub.2CGKRK
conjugated with NW inhibits HUVEC capillary-like tube formation in
vitro.
[0349] Caspase-3 activity was determined in HUVEC cells 24 h after
treatment with 3 or 10 microgram .sub.D(KLAKLAK).sub.2CGKRK-NW
using a caspase-Glo 3/7 assay kit. Two hours after reagent was
added luminescence was recorded on luminometer. FIG. 10 shows
caspase activity by HUVEC cells treated with
.sub.D(KLAKLAK).sub.2CGKRK-NW.
[0350] HUVEC cells were treated either with CREKA-NW (SEQ ID NO:92)
as control (10 .mu.g) or .sub.D(KLAKLAK).sub.2CGKRK-NW (10 .mu.g)
for 24, 48 and 72 hr. Whole cell extracts were prepared and
analyzed by Western blotting using antibodies against cleaved
caspase-3 (exp 1) or caspase 3 (exp 2).
.sub.D(KLAKLAK).sub.2CGKRK-NW increased caspase-3 activity.
[0351] HUVEC cells were untreated (control), or treated either with
CREKA-NW (10 .mu.g), or .sub.D(KLAKLAK).sub.2CGKRK-NW (10 .mu.g)
for 24 hr. Confocal microscopy images of HUVEC cells incubated with
.sub.D(KLAKLAK).sub.2CGKRK-NW showed increased cleaved caspase-3 in
comparison to untreated or CREKA-NW (SEQ ID NO:92) treated cells.
Iron oxide NW coated with 5K-PEG-FAM-labeled
.sub.D(KLAKLAK).sub.2CGKRK peptide were intravenously injected (5
mg iron per kg body weight) into mice bearing RAS-sip53 induced
brain tumors (viral injections into the right hippocampus). Six
hours later post-injection, the mice were perfused through the
heart with PBS, and the organs were collected. Tumor sections were
stained and examined by confocal microscopy.
CGKRK-.sub.D(KLAKLAK).sub.2-NW was shown to home to glioblastoma
multiforme (GBM). Mice bearing RAS-sip53 induced brain tumors
(three weeks post-injection) were intravenously injected with NW
coated with peptides through a 5-kDa polyethylene glycol spacer.
The particles were administered every other day for 14 days (5 mg
iron/kg/day, total cumulative dose 35 mg/kg). Survival was
monitored over time (n=3 per group). FIG. 11 is a diagram of the
GBM treatment with CGKRK-.sub.D(KLAKLAK).sub.2-NW nanoworms (EXP
NUMBER 1).
[0352] Mice bearing RAS-sip53 induced brain tumors (three weeks
post-injection) were intravenously injected with NW coated with
peptides through a 5-kDa polyethylene glycol spacer. The particles
were administered every other day for 14 days (5 mg iron/kg/day,
total cumulative dose 35 mg/kg). Survival was monitored over time
(n=3 per group). FIG. 12 shows GBM treatment with
CGKRK-.sub.D(KLAKLAK).sub.2-NW nanoworms (EXP NUMBER 1).
[0353] Mice bearing RAS-sip53 induced brain tumors (injection to
the right hippocampus) were intravenously injected with NW coated
with peptides through a 5-kDa polyethylene glycol spacer. The
particles alone or co-injection with iRGD were administered once a
week for 6 weeks (one weeks post-viral injection), or every other
day for two weeks and a half weeks (three weeks post-viral
injection) via tail vein injection. All mice were monitored for
luciferase signal using the IVIS system (the lentivector contains
the luciferase reporter). FIGS. 13A and 13B show GBM treatment with
CGKRK-.sub.D(KLAKLAK).sub.2-NW nanoworms (EXP NUMBER 2). Survival
of the mice is being currently recorded (n=3 per group).
[0354] Mice were bled one day before starting the treatment and one
day following the two and a half treatment course. For the groups
of mice injected every other day another blood collection was
performed two weeks after the last day of treatment. The levels of
ALT were tested in the serum of all the mice. Normal values go from
10-40 U/L. FIG. 14 shows ALT (L-Alanine-2-Oxoglutarate
Aminotransferase) levels in mice pre and post-nanoworm
treatment.
[0355] Mice bearing RAS-sip53 induced brain tumors (injection to
the right hippocampus) were intravenously injected with NW coated
with peptides through a 5-kDa polyethylene glycol spacer. Confocal
immunofluorescent analysis of frozen RAS-sip53 induced brain
tumors. One mouse from each of the indicated groups (left side) was
euthanized and frozen sections were prepared from the brain. NW
distribution after tumor therapy showed the presence of NW coated
peptides in the tumor. Confocal images of normal organs from mice
bearing RAS-sip53 induced brain tumors injected with
FAM-.sub.D(KLAKLAK).sub.2CGKRK-NW were taken. The distribution of
FAM-.sub.D(KLAKLAK).sub.2CGKRK-NW in normal organs (in the end of
the treatment) showed the presence of
FAM-.sub.D(KLAKLAK).sub.2CGKRK-NW in the kidney and spleen. The
kidney and spleen were the only non-tumor tissues that showed
significant .sub.D(KLAKLAK).sub.2CGKRK-NW fluorescence. Presence of
the chimera peptide-NW in the spleen is due to general uptake of
nanoparticles unrelated to the homing peptide and kidney is due to
cleavage of the peptide from the particle.
[0356] FIGS. 15A and 15B show the GBM treatment with
CGKRK-.sub.D(KLAKLAK).sub.2-NW nanoworms. Panel A shows a schematic
of the experiment. Mice bearing 005 brain tumor cells (10 day
post-injection) were intravenously injected with NW coated with
peptides through a 5-kDa polyethylene glycol spacer. The particles
without and co-injection with iRGD were administered every other
day for 14 days (5 mg iron/kg/day, total cumulative dose 35 mg/kg).
Panel B shows a graph of survival. Survival was monitored over time
(n=3 per group).
[0357] .sub.D(KLAKLAK).sub.2CGKRK-NW were intravenously injected
into mice bearing U87. The particles were allowed to circulate for
6 hours (the time determined in preliminary experiments to be
optimal for differential homing). The MR Image of
.sub.D(KLAKLAK).sub.2CGKRK-NW in U87 T2-weighted MR images (Fast
Spin Echo, TR=6.4 s, TE=69 ms) shows hypointense vascular signals
throughout the tumor. Nontargeted nanoworms gave no detectable
signal in these tumors after most of the nanoparticles had been
cleared from the blood.
B. Example 2
Homing, Localization, and Effect of Homing Molecule Compositions on
Glioblastoma
[0358] This example describes examples of the disclosed
tumor-homing nanoparticle conjugates, which have been constructed
based on three novel elements: (1) A tumor-homing peptide that
specifically delivers its payload to the mitochondria of tumor
endothelial cells and tumor cells; (2) conjugation of this homing
peptide with a pro-apoptotic peptide that acts on mitochondria; and
(3) coupling of the chimeric peptide onto iron oxide nanoparticles,
which greatly enhances the pro-apoptotic activity. Treatment of
glioblastoma (GBM)-bearing mice with the nanoparticles eradicated
most tumors in one GBM model and significantly delayed tumor
development in a more aggressive model. The iron oxide component of
the nanoparticles enabled imaging of the tumors. Finally,
co-injecting these theranostic particles with the tumor penetrating
peptide iRGD further enhanced the therapeutic effect.
[0359] Anti-angiogenic therapy was thought to be a promising
therapeutic strategy, particularly for highly vascularized GBM
tumors. However, these therapies have not proven effective in GMB.
The new nanosystem technology disclosed herein shows an
unprecedented efficacy in treating GBM as it eradicated most tumors
in one mouse GBM model and greatly delayed the demise of the
animals in another, more aggressive model. Both of these models had
proven completely resistant to other treatment modalities,
including anti-angiogenic agents.
[0360] Tumor blood vessels have in the recent years become an
important therapeutic target. As a tumor grows, the blood vessels
grow with it, and this growth primarily takes place through
angiogenesis (Hanahan, 1996; Alitalo and Ferrara). Therefore,
inhibiting angiogenesis has become a mainstream therapeutic
strategy in cancer treatment. The special features of tumor
vasculature also enable another strategy, homing-based (synaphic)
delivery of drugs (Ruoslahti, 2010). Tumor blood vessels express
various cell surface and extracellular matrix proteins that normal
vessels do not express or do so at much lower levels than tumor
vessels (Hanahan, 1996; Ruoslahti, 2010). These specific vascular
markers are readily available to bind circulating ligands, such as
peptides and antibodies (Allen, 2004; Jain, 1986; Ruoslahti, 2002).
Drugs attached to such ligands will become concentrated in tumor
tissue, improving efficacy and allowing the exposure of normal
tissues to be reduced (Ruoslahti, 2010).
[0361] Vascular markers can be explored in an unbiased manner by in
vivo screening of phage libraries that display random peptide
sequences (Pasqualini and Ruoslahti, 1996). This approach has
yielded a variety of homing peptides specific for tumor vasculature
and tumor cells (Arap, 1998; Laakkonen, 2002; Sugahara, 2009). The
pentapeptide CGKRK (Cys-Gly-Lys-Arg-Lys; SEQ ID NO:1) was
originally identified by in vivo phage library screening with
epidermal tumors (Hoffman, 2003). It recognizes the vessels in most
tumors and in matrigel plug angiogenesis assays (Hoffman, 2003).
CGKRK is internalized into the target cells and can take a payload
with it. Intravenously injected CGKRK into tumor mice specifically
accumulates in the tumor localizing in both endothelial cells and
tumor cells, but is not detectable in normal tissues (Hoffman,
2003). CGKRK peptide was chosen as the homing peptide for this
study because of its excellent targeting specificity, cell
internalizing properties, simple structure, and the availability of
the sulfhydryl group in the cysteine side chain for
conjugation.
[0362] The .alpha.-helical amphipathic peptide,
.sub.D[KLAKLAK].sub.2 (SEQ ID NO:3), was originally designed as a
synthetic anti-bacterial peptide that disrupts the bacterial cell
membrane, but is less toxic to eukaryotic cells (Javadpour, 1996).
However, when internalized into eukaryotic cells,
.sub.D[KLAKLAK].sub.2 disrupts the mitochondrial membrane, which is
similar to the cell membrane bacteria, initiating apoptotic cell
death (Ellerby, 1999). Conjugating .sub.D[KLAKLAK].sub.2 with
homing peptides have produced compounds with specifically
accumulate at the target of the homing peptide causing cell killing
(Ellerby, 1999; Arap, 2002; Gerlag et al.). In this example, a
tumor-homing .sub.D[KLAKLAK].sub.2 compound was made by conjugating
.sub.D[KLAKLAK].sub.2 to CGKRK.
[0363] .sub.D[KLAKLAK].sub.2 is a highly toxic compound, even when
specifically targeted to tumors (Arap et al., 2002). Administering
toxic drugs in a nanoparticle formulation can reduce toxicity.
Examples include paclitaxel-albumin nanoparticles (Abraxane.RTM.)
and doxorubicin liposomes (Doxil.RTM.), both of which are in
clinical use. Other advantages of nanoparticles include that
compounds coupled onto their surface can be presented in a
multivalent fashion, which increases the binding efficiency at the
target, and that multiple functions can be built into a
nanoparticle. These features of nanoparticles are used in the
disclosed compositions and in using the CGKRK-.sub.D[KLAKLAK].sub.2
conjugate. Iron oxide nanoworms (NWs) are useful as the
nanoparticle scaffold because, for example, iron oxide can be used
as an MRI contrast agent, making the resulting nanoparticle a
theranostic compound, a compound with both a therapeutic and
diagnostic function.
[0364] A common disadvantage of nanoparticles as drugs is that
their large size can make it more difficult for them to penetrate
from the blood into tissues than is the case with simple molecules,
limiting the effects to the vessels and their immediate vicinity.
Recently discovered tumor-penetrating peptides can be used with the
disclosed compositions to solve this problem. These peptides, an
example of which is a 9-amino acid peptide named iRGD (CRGDKGPDC or
Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys; SEQ ID NO:134). These peptides
bind to a primary receptor (.alpha..nu. integrins in the case of
iRGD), then are proteolytically processed to unmask an R/KXXRK-OH
motif which binds to neuropilin-1 activating a transport pathway
across the vessel wall and through tissue (Teesalu et al., 2009;
Sugahara et al., 2009). A payload does not have to be coupled to
the peptide to be transported; the pathway is a bulk transport
pathway that will sweep along bystander molecules and nanoparticles
(Sugahara et al., 2010). The final element in our
CGKRK-.sub.D[KLAKLAK].sub.2-nanoparticle regimen was to combine the
nanoparticles with iRGD in tumor therapy.
[0365] Glioblastoma (GBM) is the most frequent primary brain tumor
in adults and has a poor prognosis. Despite a multi-modality
treatment approach, which includes surgery, irradiation, and
chemotherapy; the median survival is only 12 months (Wen, 2008).
Thus, more effective treatments are desperately needed for this
cancer. Here we use the targeted .sub.D[KLAKLAK].sub.2
nanoparticles to treat experimental GBM tumors.
[0366] 1. Results [0367] i. CGKRK Peptide Homing to Brain Tumor and
its Co-Localization with Mitochondria in Cells
[0368] The CGKRK peptide recognizes endothelial cells and tumor
cells in various types of tumors (Hoffman, 2003). CGKRK homing to
GBM tumors (Marumoto, 2009; Soda, 2011) was tested for this
example. Intravenously injected CGKRK strongly accumulated in GBM
tumors, as indicated by the rhodamine label on the peptide, but not
in normal tissues. This peptide was used as a targeting agent for
the glioblastomas for compositions in this example.
[0369] CGKRK has the ability to become internalized into the target
cells and take a payload with it (Hoffman, 2003). To evaluate the
intracellular localization of CGKRK peptide, live cell imaging was
performed with FAM-CGKRK and found it co-localized with a
mitochondrial marker in HUVEC and U87, human glioma cells. To
determine the specificity of CGKRK to the mitochondria,
mitochondria were isolated from liver, incubated with FAM-CGKRK and
an excess of either non-labeled CGKRK or control peptide (CREKA;
SEQ ID NO:92). The specificity of the CGKRK peptide binding to the
mitochondria was competitively inhibited by unlabeled CGKRK but not
CREKA (FIG. 17). Furthermore, a phage binding assay to the isolated
mitochondria was performed and an 80 fold increase in binding of
CGKRK-phage compare to control was found (FIG. 18) indicated that
mitochondria are the primary subcellular target organelle of CGKRK
peptide. [0370] ii. CGKRK Peptide Binds to p32 Protein.
[0371] To identify the target for the CGKRK peptide in
mitochondria, CGKRK peptide coupled SulfoLink Resin was incubated
with extracts from the mitochondria purified from mouse livers,
which we have shown significantly binds CGKRK (FIGS. 17 and 18).
Bound proteins were eluted with excess of free CGKRK peptide (2 mM)
or CREKA peptide as a control. CGKRK bound a specific band below
36-kDa and was not seen in the controls. The specific band was
identified as C1qBP or p32 by mass spectrometry. The identification
of the CGKRK-binding protein as p32 was confirmed by
immunoblotting.
[0372] Saturation binding experiments gave an average binding
affinity of K.sub.d=0.2 mg/ml for the CGKRK-p32 interaction (FIG.
19). Finally, blocking purified p32 with full length antibody
against p32 reduced the binding of biotin CGKRK in a
concentration-dependent manner up to 40% (FIG. 26). These results
indicate that indeed CGKRK recognizes p32 in the mitochondria.
[0373] iii. Intratumoral Distribution of Iron Oxide Nanoworms
Coated with CGKRK.sub.D(KLAKLAK).sub.2
[0374] A variety of anti-cancer drugs show an enhanced anti-tumor
effect when they are coupled to a tumor-homing peptide (Arap, 2002;
Curnis, 2000; Ellerby, 1999; Hamzah, 2008; Karmali, 2009). It was
realized that the mitochondria localization of CGKRK provides a way
of improving the delivery of a pro-apoptotic peptide. A targeting
system was set up that consists of 3 elements: a tumor-homing
peptide (CGKRK; Hoffman, 2003), a pro-apoptotic peptide
[.sub.D[KLAKLAK].sub.2; Ellerby, 1999 (an example of a membrane
perturbing peptide)], and iron oxide nanoparticles dubbed nanoworms
(NWs) because of their elongated shape (Agemy, 2010; Park, 2009).
The two peptides were synthesized as a chimeric peptide that is
covalently linked to the NWs through a 5K-polyethylene glycol (PEG)
linker.
[0375] Intravenously injected NWs coated with the
CGKRK-.sub.D[KLAKLAK].sub.2 chimeric peptide accumulated mainly in
tumor vessels of different mouse and human GBM xenograft model
tumors (005, Human GBM spheres, and U87). The vessels of the intact
brain did not attract CGKRK-.sub.D[KLAKLAK].sub.2-NWs. NWs coated
only with CGKRK also accumulated in tumor vessels, whereas
.sub.D(KLAKLAK).sub.2-coated NWs did not. No fluorescence from the
various NW formulations was observed in normal tissues of the
tumor-bearing mice, with the exception of the liver and the spleen,
which take up all nanoparticles non-selectively.
[0376] To demonstrate use of the iron oxide component in the
targeted pro-apoptotic peptide-NW as an MRI contrast agent for
clinical applications, magnetic resonance imaging (MRI) was
performed. Magnetic resonance imaging of 005 tumors after
intravenous injection of CGKRK.sub.D[KLAKLAK].sub.2-NWs showed
hypointense vascular signals throughout the tumor. [0377] iv.
Targeted Pro-Apoptotic Peptide-NW Induces Apoptosis
[0378] To evaluate the ability of CGKRK-.sub.D[KLAKLAK].sub.2-NWs
to induce apoptosis, the co-localization of the particles with a
mitochondrial marker was assessed. CGKRK-NWs and
CGKRK-.sub.D[KLAKLAK].sub.2-NWs were taken up into the cells and
co-localized with mitochondria whereas only small amounts of
.sub.D[KLAKLAK].sub.2-NWs internalized and co-localized with
mitochondria. Next, the cell death affectivity of the peptide
compared to the targeted-NWs was evaluated. To be able to quantify
the amount of peptide that is coupled onto the NWs,
FAM-CGKRK-.sub.D[KLAKLAK].sub.2 peptide was coupled onto the NWs
via a reducible 5-kDa PEG linker. The linker was cleaved from the
NWs and the amount of peptide present on the NWs was determined by
UV-spectrophotomer using standard curve for the free FAM peptide
and used to calculate the IC50. HUVEC cells, 005 cells
trans-differentiated to cancer endothelial cells (T3) (Soda, 2011)
and U87 cells were used. Coupling the CGKRK-.sub.D[KLAKLAK].sub.2
peptide to NWs increases the cytotoxicity hundreds of times more
than the monomeric peptide.
[0379] To further analyze this cytotoxicity effect it was checked
whether CGKRK-.sub.D[KLAKLAK].sub.2-NWs can lead to cell death via
apoptosis as was previously reported for the .sub.D[KLAKLAK].sub.2
coupled to an internalizing peptide (Ellerby, 1999). Annexin V
staining confirmed that treatment with pro-apoptotic peptide-NWs
induces cell death through apoptosis (FIGS. 21 and 27). A
significant increase of apoptotic cells was observed after
treatment with CGKRK-.sub.D[KLAKLAK].sub.2-NWs and
.sub.D[KLAKLAK].sub.2-NWs in HUVEC (around 60% after 48 hr), and in
T3 cells (around 35% after 72 hr). The control particles CREKA-NWs
and CGKRK-NWs showed no significant effect. Moreover, when the
particles were washed after 30 min of incubation, only
CGKRK-.sub.D[KLAKLAK].sub.2-NWs induced significant apoptosis in
both types of cells (40-50% after 72 hr), emphasizing the important
role of CGKRK as an internalizing peptide (FIG. 22). Furthermore,
apoptotic cell death by CGKRK-.sub.D[KLAKLAK].sub.2-NWs induced
caspase-3 cleavage.
[0380] To study the role of CGKRK-.sub.D[KLAKLAK].sub.2-NW on
angiogenic blood vessels in vitro tube formation on HUVEC cells was
tested. CGKRK-.sub.D[KLAKLAK].sub.2-NWs significantly reduce the
ability of HUVEC to form tube like structure on matrigel whereas
CGKRK-NWs have no significant effect. To assess the anti-angiogenic
effect in vivo, matrigel plaque assays were employed. Matrigel/bFGF
bearing Balb/c nude mice were treated i.v. with either PBS or
CGKRK-.sub.D[KLAKLAK].sub.2-NWs. After 14 days, the mice were
perfused with cy5-lectin, and the matrigel plugs were excised.
Imaging of the matrigel plaque by near-infrared dye and confocal
microscopy revealed that treatment with
CGKRK-.sub.D[KLAKLAK].sub.2-NWs leads to significant decrease in
blood vessel formation compared to the control plaque. [0381] v.
Therapeutic Efficacy of Targeted Pro-Apoptotic Peptide-Coated NW in
Glioblastoma
[0382] Given the observations that the targeted pro-apoptotic
peptide-coated NWs are more effective in cell death than the
peptide alone and that the cell death is induced by the same
mechanism, the therapeutic effect of
CGKRK-.sub.D[KLAKLAK].sub.2-NWs in GBM was tested. Mouse
glioblastoma models were used that closely resemble human
glioblastomas in their aggressiveness and the diffuse spreading of
the tumor cells into the normal brain tissue (Marumoto, 2009). The
model was further refined by using a single lentiviral vector
expressing H-RasV12 oncogene and a siRNA targeting p53 (Soda,
2011). Mice injected with the lentivirus in the hippocampus
invariably develop glioblastomas that have a highly predictable
course of tumorigenesis that results in the death of the mice 2 to
3 months post-injection. Systemic CGKRK-.sub.D[KLAKLAK].sub.2-NWs
treatment for 3 weeks every other day cured almost all mice
injected with the H-RasV12-sip53 lentiviral vector compared with
mice that received PBS or .sub.D(KLAKLAK).sub.2-NWs alone, that
succumbed to the disease almost at the same time (FIG. 23).
Luciferase signal was monitored (the lentiviral vector contains the
luciferase reporter) starting from 6 weeks post-viral injection
(that also correlates to the end of the treatment) and at 6, 7.5,
8, 9.5, 11, and 13 weeks. Tumor was not visualized in the mice
treated with CGKRK-.sub.D[KLAKLAK].sub.2-NWs. In addition, H&E
staining showed a lack of detectable tumor tissue in a mouse
treated with CGKRK-.sub.D[KLAKLAK].sub.2-NWs compared to a control
mouse that has a relative big tumor at the end of the treatment.
Histological analysis of the CGKRK-.sub.D[KLAKLAK].sub.2-NW treated
tumors at the end of the study showed small tumors with a lot of
particles in the blood vessels compared to non targeted
.sub.D[KLAKLAK].sub.2-NW that revealed big tumors and no evidence
of particles in the tumor.
[0383] Toxicology analyses indicated some liver toxicity
(nanoparticles non-specifically accumulate in the liver) judging
from a moderate elevation in the serum level of the liver enzyme
L-alanine-2-oxoglutarate aminotransferase (FIG. 28). The values
normalized within 2 weeks after the treatment was discontinued. The
particles are not immunogenic as was determined by ELISA against
anti-Rhodamine Abs in serum of treated mice (FIG. 29A). Minor
evidence was found of macrophage activation in an IL-6 assay (FIG.
29B). Furthermore, there is no evidence of damage to the kidney by
H&E staining. These modest toxicities are in contrast with the
severe toxicity of the monovalent .sub.D[KLAKLAK].sub.2 conjugates
that have been used before (e.g., Arap, 2002).
[0384] The second model uses a tumor cell line (005), which was
originally isolated from a glioblastoma tumor induced by the
lentiviral method (Marumoto, 2009). Mice transplanted with 005
tumor cells usually die 5-6 weeks post inoculation. The tumors
retain the invasive human glioblastoma-like properties described
before (Marumoto, 2009). CGKRK-.sub.D[KLAKLAK].sub.2-NW treatment
increased the median survival time from 32 to 52 days in this
experiment (FIG. 24). Continuous treatment given in a repeated
experiment with this model did not give further benefit (FIG. 24).
Confocal microscopy at the end of the treatment confirmed that many
of the blood vessels in the tumors of the mice treated with
CGKRK-.sub.D(KLAKLAK).sub.2-NWs were filled with peptide particles.
Lectin perfusion of the 005 tumor mice at the end of the treatment
showed that the treated tumors have almost no blood vessel
perfusion, indicating that the targeted NWs have destroyed the vast
majority of the blood vessels. In U87, a human glioblastoma cell
line, the development of the tumor was also significantly delayed
(30 days) (FIG. 30). [0385] vi. Enhancement of the Tumor
Penetration and Therapeutic Efficacy of
CGKRK.sub.D(KLAKLAK).sub.2-NW by iRGD.
[0386] A peptide dubbed iRGD [sequence: CRGD(K/R)GP(D/E)C (SEQ ID
NO:4); CendR sequence underlined] enhances tumor penetration of
iRGD-bound and, surprisingly, co-administered compounds (Sugahara,
2009; Sugahara, 2010; U.S. Patent Application Publication No.
2009-0246133). This peptide comprises two active sites: an RGD
motif (Ruoslahti, 2002) and a cryptic CendR sequence RGDK (Teesalu,
2009). The RGD homing motif directs the peptide to .alpha..nu.
integrins on tumor endothelium, where the peptide is
proteolytically processed to expose the CendR motif at the
C-terminus. The activated CendR motif binds to neuropilin-1
(NRP-1), which mediates extravasation, tumor penetration, and cell
entry of the C-terminally truncated peptide (Sugahara, 2009;
Teesalu, 2009). Co-administration of iRGD with uncoupled drugs
increases the accumulation and spreading of the drug in tumor
tissue, enhancing the activity of the drug, but not the side
effects (Sugahara, 2010).
[0387] The combination of the NWs with iRGD in a 005 tumor model
(NW treatment was only partially successful) was tested in order to
determine that iRGD co-administration system can be used to enhance
the tumor penetration and therapeutic efficacy of
CGKRK-.sub.D[KLAKLAK].sub.2-NW. Non-labeled iRGD was intravenously
co-injected with CGKRK-.sub.D[KLAKLAK].sub.2-NW and confocal
microscopy analysis showed that NWs co-injected with iRGD were able
to spread into the extravascular tumor tissue compared to
co-injecting with CRGDC where the particle accumulated mainly in
tumor vessels. CGKRK-.sub.D[KLAKLAK].sub.2-NWs co-injected with
iRGD treatment increased the median survival time from about 50
days to greater than 80 days (FIG. 25).
[0388] 2. Discussion
[0389] The peptide used in this example was shown to have specific
affinity for mitochondria. Several lines of evidence show that
CGKRK has the ability to take a payload to the mitochondria. First,
live cell imaging shows colocalization with the mitochondria marker
than phage binding assay and inhibition assay to purified
mitochondria from mouse liver show the specificity of CGKRK to
mitochondria. Second, pull-down assays of mitochondria extracts
with CGKRK peptide revealed a specific band identified by mass
spectrometry as p32. The p32 protein, a homotrimer in solution and
solid state, is primarily mitochondrial, but it can be found in the
cytoplasm, nuclei, and at the cell surface (Ghebrehiwet, 1994;
Braun, 2000; Dedio, 1996; Kittlesen, 2000; Mahdi, 2002; Mahdi,
2001). The tumor homing peptide Lyp-1 targets also p32 (Fogal,
2008). CGKRK and Lyp-1 (CGNKRTRGC; SEQ ID NO:127) could share the
same binding site on p32. The affinity binding of CGKRK is 15 time
higher then Lyp-1 (Fogal, 2008), which may be attributable to its
size and linear structure which due to the decreased steric cloud
at each of the three binding sites of the trimeric protein.
[0390] Many reports previously describe the conjugation of the
pro-apototic peptide, .sub.D[KLAKLAK].sub.2, to different cell
penetrating peptide (Arap, 2002; Fantin, 2005; Karjalainen, 2011;
Mai, 2001; Rege, 2007), antibody fragment (Marks, 2005; Rege,
2007), or encapsulation into nanostructures (Ko, 2009; Standley,
2010) to target special types of tumor. Other sequence
modifications introducing more hydrophobic residues lead to
increase internalization and the toxicity (Horton, 2009). The main
limitation of this treatment is the high dose of
.sub.D[KLAKLAK].sub.2 needed causes kidney toxicity presumably by
the non-proteolysable D residues in the peptide (Arap, 2002;
Karjalainen, 2011). In this study the CGKRK-.sub.D[KLAKLAK].sub.2
peptide was coupled to NWs, which creates a multifunctional display
of the peptide and makes the peptide hundreds of times more
effective than the monomeric peptide (NWs LC.sub.50 value was
0.05-0.15 .mu.M compare with the free peptide 14-25 .mu.M) (Table
3). An untargeted multivalent display of the .sub.D(KLAKLAK).sub.2
on nanoparticles was reported to enhance in vitro internalization
of the nanoparticles into cells (Standley, 2010) while the in vivo
effects were not studied. Furthermore, results from histopathology
and blood toxicity assays after tumor treatment indicated that the
NWs do not elicit any apparent toxicity or negative health
effects.
[0391] This example shows that CGKRK-.sub.D[KLAKLAK].sub.2-NWs
induce cell death by apoptosis in the same mechanism as the peptide
conjugate .sub.D[KLAKLAK].sub.2 alone (Ellerby, 1999). Consistent
with the cell viability results, CGKRK-.sub.D[KLAKLAK].sub.2-NWs
and .sub.D[KLAKLAK].sub.2-NWs induced Annexin expression whereas
CGKRK-NWs did not (FIG. 21). Washing the cells a short time after
the particle incubation highlights the targeting efficacy of the
moiety in this chimera. The cell death by
CGKRK-.sub.D[KLAKLAK].sub.2-NWs was caspase dependent obtained
through pro-caspase 3 processing. Apoptosis in proliferating
endothelial cells, cancer endothelial cells and GBM cell line in
vitro cells treated with the CGKRK-.sub.D[KLAKLAK].sub.2-NWs
indicate that CGKRK mediated delivery into the cells and subsequent
internalization into mitochondria then .sub.D[KLAKLAK].sub.2 motif
disturbs mitochondrial membrane to cause cell death.
[0392] CGKRK-NWs home to blood vessel in prostate cancer (Agemy,
2010). Glioblastomas are generally highly angiogenic tumors (Chi,
2009; Jain, 2007) thereby serves as an ideal model for this
therapy. In the current study it was found that CGKRK peptide shows
significant homing to GBM tumor models and CGKRK-NWs home to GBM
tumor blood vessels. Conjugation of CGKRK-.sub.D[KLAKLAK].sub.2 to
iron oxide retained its biological properties in different types of
GBM including a human model. This example shows that
CGKRK-.sub.D[KLAKLAK].sub.2-NWs are a good contrast agent for brain
glioma and could be used to selectively improve the detection of
tumor with MRI in vivo.
[0393] 3. Experimental Procedures
[0394] Cell lines and tumors. Human umbilical vein endothelial
cells (HUVEC; Lonza Walkersville, Walkersville, Md.) were cultured
using EBM-2 medium with endothelial cell growth supplement (Lonza
Walkersville, Walkersville, Md.). Human astrocytoma cell line (U87)
was grown in Dulbecco's modified Eagle's medium (DMEM) plus 10%
fetal bovine serum and 1% glutamine pen-strep (Invitrogen,
Auckland, NZ). Mouse GBM-initiating 005 cell line was established
as described (Marumoto, 2009). The 005 cells were maintained in N2
medium, which contains DMEM/F-12 (Omega Scientific), 1% N2
supplement (Invitrogen), 20 ng/mL human FGF-2 (Prepotech), 20 ng/mL
human EGF (Promega, Madison, Wis.), and 40 .mu.g/mL heparin
(Sigma-Aldrich, St. Louis, Mo.). T3 cells were obtained by
differentiation induction of 005 cells cultured in EGM-2 (Lonza
Walkersville, Walkersville, Md.). Human GBM spheres were obtained
and cultured as described previously (Soda, 2011). Mouse
GBM-initiating 005 cells were transplanted into brains of NOD-SCID
mice. A total of 3.times.10.sup.5 cells were suspended in 1.5 .mu.l
of PBS and injected stereotaxically in the right hippocampus. Human
GBM spheres xenografts were created by injecting 0.5.times.10.sup.6
cells orthotopically into NOD-SCID mice in 1.5 .mu.l of PBS. Animal
experimentation was performed according to procedures approved by
the Animal Research Committee at the University of California,
Santa Barbara, The Sanford-Burnham Medical Research Institute, and
The Salk Institute for Biological Research, San Diego.
[0395] Peptide synthesis. Peptides were synthesized with an
automatic microwave assisted peptide synthesizer (Liberty; CEM,
Matthews, N.C.) using standard solid-phase Fmoc/t-Bu chemistry with
2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate methanaminium (Anaspec, Inc., San Jose, Calif.)
as the coupling reagent. During synthesis, the peptides were
labeled with 5(6)-carboxyfluorescein (FAM) (Sigma-Aldrich, St.
Louis, Mo.) with a 6-aminohexanoic acid spacer separating the dye
from the sequence. The peptides were cleaved from the resin using
95% trifluoroacetic acid (Sigma-Aldrich, St. Louis, Mo.) with 2.5%
water and tri-isiopropylsilane (Sigma-Aldrich, St. Louis, Mo.).
Subsequent purification by High Performance Liquid Chromatography
(Gilson Inc., Middleton, Wis.) gave peptides with >90%
purity.
[0396] Isolation of mitochondria and peptide/phage binding.
Mitochondria were isolated from livers of Balb/c mice using
differential centrifugation with buffers from a Pierce
mitochondrial isolation kit for tissue according to the
manufacture's instruction (Pierce Biotechnology, Rockford, Ill.).
To test the binding of the CGKRK peptide to mitochondria, purified
mitochondria were pre-incubated with various concentrations of
non-labeled CGKRK or control peptide (CREKA; SEQ ID NO:92) for 30
min 4.degree. C. FAM-CGKRK was then added and incubated for an
additional 1 hour. The binding of the FAM peptide was quantified by
fluorescence. In phage binding assays, purified mitochondria were
suspended in 10 ml DMEM supplemented with 1% BSA, and incubated
with 5.times.10.sup.8 plaque-forming units (pfu) of
peptide-displaying phage, overnight at 4.degree. C. The
mitochondria were washed 3 times with DMEM/BSA, the phage were
collected with lysogeny broth containing 1% NP-40, and quantified
by plaque assay.
[0397] Affinity chromatography. Mitochondria were lysed in PBS
containing 400 mM n-octyl-beta-D-glucopyranoside (Calbiochem, La
Jolla, Calif.), and clarified lysates were incubated with
CGKRK-coated Sulfolink-beads (Pierce biotechnology, Rockford,
Ill.). After washing, bound proteins were eluted with lysis buffer
containing 2 mM free CGKRK peptide and separated by SDS-PAGE. Gel
bands excised from silver-stained gels were analyzed by MALDI-TOF
mass spectrometry at the Burnham Institute for Medical Research
Proteomics Resource.
[0398] Affinity measurements. The affinity of CGKRK for p32 was
measured by an ELISA-based assay. Wells in 96-well plates were
coated with 3 .mu.g/ml of purified p32 protein and incubated for 1
hour at 37.degree. C. with various concentrations of biotinylated
LyP-1 peptide in PBS (100 .mu.l/well). After washing with TBS
containing 1 mmol/l CaCl.sub.2 and 0.01% Tween 20,
streptavidin-conjugated horseradish peroxidase (Zymed, San
Francisco, Calif.) was added to the wells and incubated for 1 hour
at room temperature. Peptide binding to p32 was quantified with
2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(Sigma-Aldrich, St. Louis, Mo.) as substrate. Wells without p32
coating were used to determine background binding. Kd values were
calculated using Prism software.
[0399] Cell proliferation assay and imaging. The MTT
[3-[4,5-dimethylthazol-2-yl]-2,5-diphenyl tetrazolium-bromide]
assay (Molecular Probes, Eugene, Oreg.) was used to quantify cell
proliferation. Cells were seeded in complete medium into 96-well
plates (5.times.10.sup.3 cells/well), and allowed to attach
overnight at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere.
The culture media was then removed and various concentrations of
peptides or NWs were added. After 48, and 72 hours, 10 .mu.L of the
MTT reagent (5 mg/mL in PBS) was added to each well. The medium was
removed from cells after 3 hours, and 100 ml of DMSO:MEOH (1:1 v/v)
were added to each well. The plates were read at a wavelength of
595 nM. For live cell imaging, cells seeded on glass-bottom plates
(Willco, Amsterdam, Netherlands), and 24 hours later, the cells
were washed and incubated with FAM-labeled peptides or NWs for 45
min at 37.degree. C. The cells were then rinsed three times with
PBS and incubated for an additional 15 min at 37.degree. C. with
500 nm MitoTracker Red (Molecular Probes, Eugene, Oreg.) followed
by nuclear staining with Hoechst 33342 DNA dyes (Molecular Probe,
Eugene, Oreg.) for 12 min. The cells were analyzed with Fluoview FV
500 confocal microscopy (Olympus America, Center Valley, Pa.).
[0400] Immunoblot analysis of NW-bound proteins. HUVEC cells were
incubated 24 and 48 hr with 10 .mu.g/ml
CGKRK.sub.D[KLAKLAK].sub.2-NWs or CREKA-NW and lysed with RIPA
buffer (Pierce, Rockford, Ill.) according to the manufacturer's
instructions. The lysates were separated by SDS-PAGE. After
transfer of the proteins onto nitrocellulose membranes for 2 hours
at 200 mA, the membrane was treated for 1 hour at room temperature
with TBS-0.05% Tween containing 5% milk, incubated with 1 mg/ml
anti-caspase 3 (Cell Signaling, Denvers, Mass.) and anti-p32
(R&D system, Minneapolis, Minn.), followed by anti-rabbit IgG,
HRP-linked antibody (Cell Signaling, Denvers, Mass.).
[0401] Flow cytometry. Cells were harvested and stained using the
Annexin V-PE apoptosis detection kit (BD Pharmingen) and analyzed
on a BD LSR II flow cytometer (Becton Dickinson).
[0402] In vivo matrigel angiogenesis. Two-month old Balb/c nu/nu
mice were subcutaneously injected bilaterally in the inguinal area
with 500 ml of matrigel (Becton Dickson, Bedford, Mass.) with or
without of 500 ng recombinant human bFGF (Becton Dickson, Bedford,
Mass.) as an angiogenesis stimulant. The mice were treated every
other day with PBS or CGKRK-.sub.D[KLAKLAK].sub.2-NWs (5 mg/kg) for
2 weeks. At the end of the treatment, the mice were sacrificed
under anesthesia by perfusion through the heart with far-red
fluorescent Alexa Fluor 647 isolectin GS-IB4 conjugate (Molecular
Probes, Eugene, Oreg.), followed by further perfusion with
.about.10 ml of 4% PFA. The matrigel plugs were imaged by the
Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln,
Nebr.), and for histological analyses, 7-10 .mu.m sections were cut
and viewed under a Fluoview 500 confocal microscope (Olympus
America, Center Valley, Pa.). The treatment and PBS control groups
consisted of two mice (four plugs) in each of three independent
experiments.
[0403] Tube formation assay. Wells in 24-well plates were coated
with 250 .mu.l matrigel (Becton Dickison, Bedford, Mass.). HUVECs
were detached with trypsin (Lonza Walkersville), washed with PBS,
and 10.sup.5 cells in EBM2 media with supplement as mentioned above
were subsequently plated on top of the matrigel in the presence of
5 or 10 .mu.g/ml of NWs. The cells were incubated at 37.degree. C.
for 24 hours and photographed under a bright field microscope at
40.times. magnification (Leica Microsystems, Wetzlar, Germany).
[0404] In vivo peptide homing. Mice with orthotopic brain cancers
induced as described above were used when they showed symptoms of
the presence of a tumor mass. Rhodamine-labeled CGKRK (200 .mu.g)
was intravenously injected into the mice and allowed to circulate
for 3 hours. The mice were perfused with PBS through the heart
under anesthesia, and tissues were collected and processed for
fluorescence analysis.
[0405] Preparation of NWs. NWs coated with peptides were prepared
as described (Agemy, 2010; Park, 2009). Aminated nanoworms were
pegylated with maleimide-5KPEG-NHS (JenkemTechnology, City, China).
The aminated nanoworms were pegylated with maleimide-5KPEG-NHS
(JenkemTechnology, China). Peptides were conjugated to the
nanoparticles through a thioether bond between the cysteine thiol
from the peptide sequence and the maleimide on the functionalized
particles.
[0406] Quantification peptide on NWs. The aminated nanoworms were
pegylated with OPSS-5KPEG-NHS (JenkemTechnology, China). Peptides
were conjugated to the nanoparticles through a disulfide bond
between the cysteine thiol from the peptide sequence and the
pyridyl sulfenyl protected thiol on the functionalized particles.
The CGKRK-.sub.D[KLAKLAK].sub.2-NWs linked covalently through the
disulfide linkage were treated with DTT. The concentration of free
peptide thus obtained in the solution was estimated using
fluorescence spectroscopy with a peptide standard curve.
[0407] In vivo NW injections. Mice bearing orthotopic GBM tumors
were injected into the tail vein with NWs (5 mg of iron per kg body
weight). In homing experiments, the mice were euthanized 5-6 hours
after the injection by cardiac perfusion with PBS under anesthesia,
and organs were dissected and analyzed for NWs. In tumor treatment
experiments, tumor mice were intravenously injected with NWs in 150
.mu.l PBS, or PBS as a control every other day for 3 weeks. Mice
with 005 tumors were also intravenously injected with
CGKRK-.sub.D[KLAKLAK].sub.2-NW (5 mg of iron/kg) in combination
with 4 mmol/kg of either the tumor-penetrating peptide, iRGD, or
CRGDC (SEQ ID NO:136) as a control. At the end of the treatment, 2
mice per group were euthanized and the rest of the mice were
monitored until the animal facility staff determined that a mouse's
symptoms required euthanasia.
[0408] Histology and immunohistology. Tissues were fixed in 4%
paraformaldehyde overnight at 4.degree. C., cryo-protected in 30%
sucrose overnight and frozen in OCT embedding medium. Tissue
sections (7 .mu.m) were cut and H&E stained or processed for
immunostaining. To stain for CD31, sections were first incubated
for 1 hour at room temperature with 10% serum from the species in
which the secondary antibody was generated, followed by incubation
with monoclonal anti-mouse CD31 (10 mg/ml; BD Pharmingen, San Jose,
Calif.), and Alexa 647 goat anti-rat secondary antibody (1:1000;
Molecular Probes, Eugene, Oreg.). Each staining experiment included
sections stained with the secondary antibody only as a negative
control. Nuclei were counterstained with DAPI (5 mg/mL; Molecular
Probes). The sections were mounted in Gel/Mount mounting medium
(Biomeda, Foster City, Calif.) and viewed under a Fluoview 500
confocal microscope (Olympus America, Center Valley, Pa.).
[0409] Biophotonic tumor imaging. Mice bearing luciferase-labeled
GBM tumors received injections of 3 mg per mouse of freshly
prepared luciferin substrate (Promega, Madison, Wis.) suspended in
PBS. The mice were then anesthetized with isofluorane and imaged
using the Xenogen IVIS.RTM. 100 Imaging System (Xenogen, Caliper
LifeSciences), 10 minutes post intraperitoneal injection of
luciferin at a 1-minute acquisition time in a small binning
mode.
[0410] NW toxicity studies. Serum was collected from mice before
treatment, one day after the treatment ended, and two weeks after
the treatment was concluded. ALT levels in serum were determined
using ALT (GPT) Reagent (Infinity.TM.) following the manufacturer's
instructions. The same serum samples were used to determine the
levels of IL-6 using the Mouse IL-6 ELISA Set (BD OptEIA.TM., BD
Biosciences).
[0411] Magnetic resonance imaging. Mice bearing orthotopic 005
tumors were intravenously injected with
CGKRK-.sub.D[KLAKLAK].sub.2-NW (5 mg of iron/kg). Approximately 5
hours after the NW injection, the mice were anesthetized with
isoflurane and subjected to T2* weighted MRI using the following
conditions: 3D spoiled gradient echo, TR=40 ms, TE=18.6 ms, Flip
angle=15 deg, bandwidth=115 Hz/pixel, resolution: (0.16 mm).sub.2
in plane and 0.3 mm slice thickness). Three slices averaged for
improved signal-to-noise). The instrument was Sigma HDx 3 T scanner
(GE Healthcare, Milwaukee, Wis.). After imaging, tissues of
interest were harvested and processed for H&E staining.
[0412] Statistical analysis. Data were analyzed by two-tailed
Student's unpaired t-test. P values of less than 0.05 were
considered statistically significant.
[0413] Glioblastomas are generally highly angiogenic tumors, and
VEGF is produced in high levels by the tumor cells (Chi, 2009;
Jain, 2007). Therefore, anti-angiogenic therapy was thought to be a
promising therapeutic strategy particular for glioma (Jain, 2007).
The best known antiangiogenic agents are inhibitors of VEGF-A,
notably, bevacizumab, a neutralizing antibody to VEGF-A,
demonstrating good anti-glioma activity in preclinical study, but
only marginal effect in the clinic. Combining angiogenesis
inhibitors with other anti cancer agents showed better therapeutic
effect. For example, in a phase II clinical trial, more than half
of the patients with GBM responded to the combination treatment of
anti-VEGF antibody bevacizumab and irinotecan, but this effect was
transient in most patients (Vredenburgh, 2007). Mechanisms proposed
to explain resistance to anti-VEGF therapy include activation of
other proangiogenic signaling pathways, recruitment of bone marrow
(BM)-derived myeloid cells that protect and nurture vascular cells,
protection of blood vessels by increased pericyte coverage, and
increased tumor invasion (Bergers, 2008; Shojaei, 2008). Recent
studies have shown that lack of VEGF-A receptor (VEGFR2) and bFGF
receptor by transdifferentiation of glioblastoma cells into
endothelial cells are potentially the main contribution for this
resistance (Soda, 2011).
[0414] 4. Results [0415] i. Homing of CGKRK Peptide to Glioblastoma
(GMB) Tumors and Interaction of CGKRK Peptide with
Mitochondria.
[0416] Mice bearing 005 glioma tumors in the right hippocampus were
intravenously injected with 200 .mu.g of CGKRK peptide labeled with
rhodamine. After 3 hours, the mice were perfused through the heart
with PBS, and the tumor and normal brain tissue were collected. The
inset in last panel shows section of normal brain tissue. CGKRK
peptides congregate in tumor blood vessels. Proliferating human
endothelial cells (human umbilical vein endothelial cells (HUVEC);
resembling angiogenic endothelial cells) and U87 cells were
incubated with FAM-CGKRK peptide and MitoTracker and examined by
fluorescent microscopy to assess whether the CGKRK peptide targets
mitochondria. The coincidence of the two labels showed that CGKRK
peptides co-localized with mitochondria. FAM-CGKRK was incubated
with purified mitochondria in the presence of increasing
concentrations of either unlabeled CGKRK or an unrelated peptide
(CREKA) as a control (FIG. 17). Binding of the labeled CGKRK
peptide declined in proportion to the amount of unlabeled CGKRK
peptide but not in the case of the CREKA peptide, indicating that
the binding to mitochondria by the CGKRK peptide is specific. CGKRK
phage and CREKA phage (as a control) were incubated with purified
mitochondria (FIG. 18). Titration of bound phage shows about 80
times more binding of the CGKRK phage than the control. [0417] ii.
CGKRK Peptide Binds to p32 Protein in Mitochondrial Extracts.
[0418] Proteins were extracted from mitochondria purified from
mouse livers and fractionated by affinity chromatography on CGKRK
peptide coupled SulfoLink Resin. Bound proteins were eluted with
free CGKRK peptide (2 mM), or CREKA peptide as a control. p32
protein eluted rapidly from the affinity matrix with the CGKRK
peptide but did not elute with the control CREKA peptide. This
indicates that the CGKRK peptide binds specifically to the p32
protein. An anti-p32 immunoblot of elution samples was performed.
This confirmed that the eluted protein corresponds to the p32
protein. Binding of increasing amounts of biotin-labeled CGKRK
peptide to immobilized p32 protein was detected with streptavidin
coupled to horseradish peroxidase and normalized to nonspecific
binding in the absence of p32 (FIG. 19). The affinity of the
peptide for p32 calculated from the binding curves is
Kd=0.2.+-.0.068 .mu.g/ml. Percent of inhibition was assessed using
binding inhibition curves in the presence of increasing
concentrations of non-labeled CGKRK peptide or LyP-1 peptide (FIG.
20). Inhibition of binding drops much more quickly for both
peptides in the presence of non-labeled CGKRK than for non-labeled
LyP-1 peptide. [0419] iii. Homing of
CGKRK.sub.D[KLAKLAK].sub.2-Nanoworms (NWs) to GMB Tumors.
[0420] A chimeric peptide consisting of a tumor-homing peptide
(CGKRK) and a pro-apoptotic peptide (.sub.D[KLAKLAK].sub.2) was
covalently coupled to iron oxide nanoparticles (NWs; length 80-100
nm, width 30 nm; FIG. 16). Iron oxide NWs coated with Rd-labeled
CGKRK-.sub.D[KLAKLAK].sub.2 peptide (through a 5K-PEG linker) were
intravenously injected (5 mg iron per kg body weight) into mice
bearing either 005 tumors, or xenograft tumors generated with human
GBM spheres or U87 cells. The tumor cells were injected into the
right hippocampus. Five to six hours after the injection, the mice
were perfused through the heart with PBS, and the organs were
collected. Tumor sections were stained and examined by confocal
microscopy. The Rd-labeled CGKRK-.sub.D[KLAKLAK].sub.2-coated
particles, tumor cells (both the human GBM spheres and U87 cells
expressed green fluorescent protein), blood vessels stained with
anti-CD31, and nuclei stained with DAPI were visualized. CGKRK
peptides congregate in tumor blood vessels for all three tumor
types. Rd-labeled CGKRK.sub.D[KLAKLAK].sub.2-NWs were intravenously
injected into tumor-bearing mice and tumors visualized by T2*
weighted MRI (3D spoiled gradient echo. Signal from the nanoworm
compositions coincided with the tumor location. [0421] iv.
.sub.D[KLAKLA].sub.2CGKRK-NW Conjugates Internalize Into Activated
Endothelial Cells, Co-Localize with Mitochondria, and Induce Cell
Death by Apoptosis.
[0422] Live HUVEC were incubated for 2 hours at 37.degree. C. in
the presence of fluorescein (FAM)-labeled NWs (CGKRK peptide,
.sub.D[KLAKLAK].sub.2 peptide, or CGKRK.sub.D[KLAKLAK].sub.2
peptide and for 15 minutes in the presence of a marker for
mitochondria (MitoTracker). DNA was counterstained with Hoechst
33342. The cells were visualized by confocal microscopy. Only the
CGKRK.sub.D[KLAKLAK].sub.2 peptide showed extensive localization in
mitochondria. FAM-CGKRK.sub.D[KLAKLAK].sub.2 peptide was coupled
onto the NWs via a reducible 5-kDa PEG linker. The linker was
cleaved from the NWs using DTT, and the amount of peptide present
on the NWs was determined by fluorescence measurements in solution
(to circumvent quenching on the NW surface) and used to calculate
IC50 (Table 3).
TABLE-US-00003 TABLE 3 IC50 Cell Line Peptide (.mu.M) Peptide on NW
Fold Increase HUVEC 14 0.05 280.0 T3 25 0.15 166.7 U87 16 0.15
106.7
[0423] HUVEC and T3 cells were left untreated (Control) or treated
with a concentration of 10 .mu.g/ml of NWs coated with either a
control peptide (CREKA), .sub.D[KLAKLAK].sub.2, or
CGKRK.sub.D[KLAKLAK].sub.2 for 24, 48 and 72 hours (FIG. 21) or the
particles were washed away after 30 min and the incubation was
continued for 72 hrs (FIG. 22). The cells were stained with Annexin
and analyzed by flow cytometry. The total percentage of
Annexin-positive cells (apoptotic and dead cells) is indicated in
FIGS. 21 and 22. Whole cell extracts (HUVEC) from the experiment in
FIG. 21 were prepared and analyzed by immunoblotting using
antibodies against cleaved caspase-3, and .beta.-actin as loading
control and also by confocal microscopy with cleaved caspase-3,
tubulin, and nuclei stained. Caspase-3 showed cleavage only in the
cells incubated with CGKRK.sub.D[KLAKLAK].sub.2 nanoworms. [0424]
v. CGKRK.sub.D[KLAKLAK].sub.2-NW Treatment of Tumors Induced by
Lentiviral Injection.
[0425] Mice bearing lenti-viral (H-RasV12-sip53) induced brain
tumors in the right hippocampus were intravenously injected with NW
coated with peptides. The particles were administered every other
day for 18 days, starting 3 weeks post-viral injection. FIG. 23
shows the survival curves of the non-treated (control) mice and for
mice treated with .sub.D[KLAKLAK].sub.2 nanoworms or
CGKRK.sub.D[KLAKLAK].sub.2 nanoworms. CGKRK.sub.D[KLAKLAK].sub.2
nanoworms provide a dramatic increase in survival compared to the
control and the .sub.D[KLAKLAK].sub.2 nanoworms. The mice were
monitored for luciferase signal using the IVIS system (the
lentiviral vector contains the luciferase reporter). Only the
control mice had detectable luciferase signal. After H&E
staining at the end of the treatment, cancer was visible at the
lentiviral injection site only in control mice. Confocal microscopy
images of brain sections from a representative mice at the end of
the treatment showed a small residual tumor in the with
CGKRK.sub.D[KLAKLAK].sub.2-NW-treated mouse but significantly more
tumor cells in the .sub.D[KLAKLAK].sub.2-NW-treated mouse. [0426]
vi. Treatment of Transplanted GBM Tumors with
CGKRK.sub.D[KLAKLAK].sub.2-NWs.
[0427] Tumors were developed by transplanting 3.times.10.sup.5 005
cells into the right hippocampus of NOD-SCID mice. Ten days
post-tumor cell transplantation, the mice were intravenously
injected with NWs. The NWs (5 mg of iron/kg) were administered
every other day for 3 weeks or administered non-stop for the same
period of time (n=8 per group). A, FIG. 24 shows survival curves of
mice treated with .sub.D[KLAKLAK].sub.2-NWs, CGKRK-NWs, and
CGKRK.sub.D[KLAKLAK].sub.2-NWs. CGKRK.sub.D[KLAKLAK].sub.2-NWs
exhibited longer survival whether administered every other day or
non-stop. Brain sections of representative mice at the end of the
treatment were stained with anti-CD31 and DAPI. The
CGKRK.sub.D[KLAKLAK].sub.2-NWs and .sub.D[KLAKLAK].sub.2-NWs were
labeled with rhodamine The tumor cells expressed green fluorescent
protein. CGKRK.sub.D[KLAKLAK].sub.2-NWs homed to tumor blood
vessels. .sub.D[KLAKLAK].sub.2-NWs did not home to tumors. Lectin
was perfused into representative tumor mice at the end of the
treatment. Vessels were stained by perfusion of biotinylated
Lycopersicon esculentum lectin and visualized by confocal
microscopy using anti-biotin. Mice treated with
CGKRK.sub.D[KLAKLAK].sub.2-NWs showed little tumor vessel labeling
and less tumor cell labeling compared to the control mice
indicating reduction in tumors and tumor vessels. [0428] vii.
Enhanced Anti-Tumor Effect of CGKRK.sub.D[KLAKLAK].sub.2-NWs
Co-Injected with iRGD.
[0429] Mice bearing orthotopic 005 tumors were intravenously
injected with CGKRK.sub.D[KLAKLAK].sub.2-NW (5 mg of iron/kg) in
combination with 4 mmol/kg of either non-labeled CRGDC or iRGD
peptide. The tumors and tissues were collected 5-6 hours later, and
analyzed by confocal microscopy. CGKRK.sub.D[KLAKLAK].sub.2-NWs in
mice coinjected with CRGDC mostly coincided with tumor blood
vessels, indicating homing to tumor blood vessels.
CGKRK.sub.D[KLAKLAK].sub.2-NWs in mice coinjected with iRGD were
localized within the tumor away form the tumor blood vessels
indicating increase cell internalization and tissue penetration.
Mice bearing orthotopic 005 tumors implanted 10 days earlier
received every other day for 3 weeks intravenous injections of
either CGKRK.sub.D[KLAKLAK].sub.2-NWs (5 mg of iron/kg) mixed with
4 mmol/kg of cRGD or iRGD. FIG. 25 shows survival curves are shown
(n=8-10 per group). Mice treated with
CGKRK.sub.D[KLAKLAK].sub.2-NWs showed a clear increase in survival
time compared to control mice and mice treated with iRGD alone,
with mice treated with CGKRK.sub.D[KLAKLAK].sub.2-NWs and iRGD
together showing the longest survival times (80% of the mice still
alive after 80 days). [0430] viii. CGKRK Peptide and
CGKRK.sub.D[KLAKLAK].sub.2-NWs in Normal Tissue of Tumor-Bearing
Mice.
[0431] Mice bearing 005 tumors in the right hippocampus were
intravenously injected with 200 .mu.g of Rd-labeled CGKRK peptide.
After 3 hours, the mice were perfused through the heart with PBS,
and tissues were collected, sectioned and analyzed for rhodamine
fluorescence. Significant CGKRK peptide appeared in kidney but not
in heart, pancreas, liver, lung, or spleen. Rd-labeled
CGKRK.sub.D[KLAKLAK].sub.2-NWs were intravenously injected (5 mg
iron per kg body weight) into mice bearing 005 tumors. The tumors
were generated by injecting 005 tumor cells into the right
hippocampus. Five to six hours after the injection, the mice were
perfused through the heart with PBS, and tissues were collected.
Tumor sections were stained with antibodies and examined by
confocal microscopy. Significant CGKRK.sub.D[KLAKLAK].sub.2-NWs
appeared in kidney but not in pancreas, liver, lung, spleen, or
normal brain. The label also appeared in urine of the mice. [0432]
ix. Inhibition of CGKRK Peptide Binding to p32 by Anti-p32.
[0433] Biotin-CGKRK at 1 .mu.g/ml was incubated in microtiter wells
coated with purified p32, and the binding was detected with
streptavidin coupled to horseradish peroxidase and normalized to
nonspecific binding in the absence of p32. The anti-32 antibody was
prepared against the full-length p32 protein (Protein Production
and Analysis Facility of the Sanford-Burnham Medical Research
Institute). The experiments were performed in triplicate; one of
two experiments with similar results is shown in FIG. 26. The
result show that the CGKRK peptide binds to p32. [0434] x. Homing
of CGKRK-NWs to GMB Tumors.
[0435] Iron oxide NWs coated with Rd-labeled CGKRK or
.sub.D[KLAKLAK].sub.2 peptide through a 5K-PEG linker were
intravenously injected (5 mg iron per kg body weight) into mice
bearing 005 tumors. The tumors were generated by injecting 005
tumor cells into the right hippocampus. Five to six hours after the
injection, the mice were perfused through the heart with PBS, and
tissues were collected. Tumor sections were stained with antibodies
and examined by confocal microscopy. The CGKRK-NWs homed to blood
vessels in the tumors. The .sub.D[KLAKLAK].sub.2-NWs did not
collect in the tumors. [0436] xi. CGKRK.sub.D[KLAKLAK].sub.2-NW
Conjugates Induce Cell Death by Apoptosis.
[0437] HUVEC and T3 cells were left untreated (Control) or treated
with 10 .mu.g/ml of NWs coated with CGKRK.sub.D[KLAKLAK].sub.2-NWs
for 48 or 72 hours. Representative results for these HUVEC and T3
cells indicating the percentage of Annexin-positive cells
(apoptotic and dead cells) are shown in FIGS. 27A and 27B,
respectively. HUVEC and T3 cells were also incubated with
CGKRK-NWs, CREKA-NWs, .sub.D[KLAKLAK].sub.2-NWs, or
CGKRK.sub.D[KLAKLAK].sub.2-NWs. The cells were washed after 30
minutes to remove excess NWs and then incubated for 72 hours.
Representative results for these HUVEC and T3 cells indicating the
percentage of Annexin-positive cells are shown in FIG. 27C. Annexin
staining and analysis by flow cytometry were used to measure
apoptosis in the cultures. [0438] xii.
CGKRK.sub.D[KLAKLAK].sub.2-NW Conjugates Inhibit In Vitro and In
Vivo Angiogenesis.
[0439] Tube formation assays were performed using primary HUVEC
plated on growth factor reduced matrigel in 5% FCS medium alone
(Control) or containing CGKRK-NWs or
CGKRK.sub.D[KLAKLAK].sub.2-NWs. The formation of networks of
capillary-like structures was viewed by phase contrast-microscopy
at 40.times. magnification 24 hours after plating. Capillary
formation was disrupted in cells treated with
CGKRK.sub.D[KLAKLAK].sub.2-NWs but not in cells treated with
CGKRK-NWs. Matrigel plugs with or without bFGF were subcutaneously
injected into Balb/c nu/nu mice. The mice were treated every other
day with intravenous injections of either
CGKRK.sub.D[KLAKLAK].sub.2-NWs (5 mg/kg) or PBS. The mice were
euthanized 14 days later, and perfused with 647 Alexa iso-lectin.
The matrigel plugs were removed from the mice and viewed
macroscopically under fluorescent light or by confocal microscopy
analysis of sections of the plugs. Representative mice treated with
matrigel and without bFGF and mice treated with matrigel, bFGF, and
CGKRK.sub.D[KLAKLAK].sub.2-NWs showed no significant vessel
formation while the control (treated with matrigel and bFGF) showed
robust vessel formation. [0440] xiii. Toxicology Analyses of Mice
Treated with CGKRK.sub.D[KLAKLAK].sub.2-NWs.
[0441] Blood L-alanine-2-oxoglutarate aminotransferase (ALT) levels
measured before (Pre-treatment), after completion of a 3-week
treatment course (After treatment) and after a subsequent 2-week
recovery period (2 weeks after treatment) are shown in FIG. 28. A
significant increase was seen in two of the mice after treatment
with CGKRK .sub.D[KLAKLAK].sub.2-NWs, but this effect had
disappeared 2 weeks after treatment. Possible active and innate
immune responses against NW was tested by measuring antibody (FIG.
29A) and IL-6 levels (FIG. 29B) in serum of mice collected as
described above. The kidneys in control mice and mice treated with
CGKRK.sub.D[KLAKLAK].sub.2-NWs were visualized after H&E
staining at the end of the treatment. No significant difference was
seen between the treatments. [0442] xiv. Treatment of Mice Bearing
Intracranial U87 Tumors with CGKRK.sub.D[KLAKLAK].sub.2-NWs.
[0443] Tumors were induced by injecting 5.times.10.sup.5
GFP-expressing U87 cells into the right hippocampus of mice.
Treatment with intravenous injections of
CGKRK-.sub.D[KLAKLAK].sub.2-NWs and control NWs was started 10 days
after the tumor cell injection and continued every other day for 3
weeks (n=5 per group). Survival curves are shown in FIG. 30.
Treatment with CGKRK-.sub.D[KLAKLAK].sub.2-NWs significantly
increased the time of survival. [0444] xv. CGKRK-Binding Proteins
in Glioblastoma Tumor Extracts.
[0445] Proteins from brain tumor extracts were bound to
insolubilized (SulfoLink Resin; Pierce Biotechnology) CGKRK
peptide. Proteins were eluted from the affinity matrix with either
CGKRK peptide or with CREKA (SEQ ID NO:92) peptide (negative
control). Silver-stained gels were used to show the proteins eluted
with CGKRK and CREKA (SEQ ID NO:92). CREKA (SEQ ID NO:92) failed to
elute any visible protein bands. CGKRK peptide eluted nardilysin
(132 Kd), nucleolin (97 Kd), cytoskeleton-associated protein 4
(p63), Hnrnpa3/Hnrnpa2b1 (39 Kd), and p32 protein (33 Kd). The
bands in the CGKRK eluate were identified by mass spectrometry as
the proteins listed.
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Moore A, Weissleder R, Bogdanov A, Jr (1997) J Magn Reson Imaging
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Biochim Biophys Acta 674: 354-371. [0468] Fernandez-Urrusuno R,
Fattal E, Rodrigues J M, Jr, Feger J, Bedossa P, Couvreur P (1996)
J Biomed Mater Res 31: 401-408. [0469] Radomski A, Jurasz P,
Alonso-Escolano D, Drews M, Morandi M, Malinski T, Radomski M W
(2005) Br J Pharmacol 146: 882-93. [0470] Khandoga A, Stampfl A,
Takenaka S, Schulz H, Radykewicz R, Kreyling W, Krombach F (2004)
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Sun G, Woods C (2005) Blood 105: 1956-1963. [0472] Gorbet M B,
Sefton M V (2004) Biomaterials 25: 5681-5703. [0473] Boccaccio C,
Sabatino G, Medico E, Girolami F, Follenzi A, Reato G, Sottile A,
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Sequence CWU 1
1
13715PRTArtificial Sequencechemically synthesized; homing
peptidePEPTIDE(1)..(5) 1Cys Gly Lys Arg Lys 1 5 26PRTArtificial
Sequencechemically synthesized; homing peptidepeptide(1)..(6) 2Cys
Arg Lys Asp Lys Cys 1 5 314PRTArtificial Sequencechemically
synthesized; membrane perturbing moleculepeptide(1)..(14) 3Lys Leu
Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 1 5 10
49PRTArtificial Sequencechemically synthesized; homing
peptideMISC_FEATURE(5)..(5)X at position 5 can be either lysine or
arginineMISC_FEATURE(8)..(8)X at position 8 can be either aspartate
or glutamate 4Cys Arg Gly Asp Xaa Gly Pro Xaa Cys 1 5
514PRTArtificial Sequencechemically synthesized; membrane
perturbing moleculepeptide(1)..(14) 5Lys Leu Ala Lys Lys Leu Ala
Lys Leu Ala Lys Lys Leu Ala 1 5 10 614PRTArtificial
Sequencechemically synthesized; membrane perturbing
moleculepeptide(1)..(14) 6Lys Ala Ala Lys Lys Ala Ala Lys Ala Ala
Lys Lys Ala Ala 1 5 10 721PRTArtificial Sequencechemically
synthesized; membrane perturbing moleculepeptide(1)..(21) 7Lys Leu
Gly Lys Lys Leu Gly Lys Leu Gly Lys Lys Leu Gly Lys Leu 1 5 10 15
Gly Lys Lys Leu Gly 20 89PRTArtificial Sequencechemically
synthesized; brain homing peptidepeptide(1)..(9) 8Cys Asn Ser Arg
Leu His Leu Arg Cys 1 5 99PRTArtificial Sequencechemically
synthesized; brain homing peptidepeptide(1)..(9) 9Cys Glu Asn Trp
Trp Gly Asp Val Cys 1 5 1021PRTArtificial Sequencechemically
synthesized; brain homing peptidepeptide(1)..(21) 10Trp Arg Cys Val
Leu Arg Glu Gly Pro Ala Gly Gly Cys Ala Trp Phe 1 5 10 15 Asn Arg
His Arg Leu 20 119PRTArtificial Sequencechemically synthesized;
brain homing peptidepeptide(1)..(9) 11Cys Leu Ser Ser Arg Leu Asp
Ala Cys 1 5 128PRTArtificial Sequencechemically synthesized; brain
homing peptidepeptide(1)..(8) 12Cys Val Leu Arg Gly Gly Arg Cys 1 5
139PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(9) 13Cys Asn Ser Arg Leu Gln Leu Arg Cys 1 5
147PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(7) 14Cys Gly Val Arg Leu Gly Cys 1 5
158PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(8) 15Cys Lys Asp Trp Gly Arg Ile Cys 1 5
168PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(8) 16Cys Leu Asp Trp Gly Arg Ile Cys 1 5
178PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(8) 17Cys Thr Arg Ile Thr Glu Ser Cys 1 5
187PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(7) 18Cys Glu Thr Leu Pro Ala Cys 1 5
198PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(8) 19Cys Arg Thr Gly Thr Leu Phe Cys 1 5
208PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(8) 20Cys Gly Arg Ser Leu Asp Ala Cys 1 5
219PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(9) 21Cys Arg His Trp Phe Asp Val Val Cys 1 5
228PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(8) 22Cys Ala Asn Ala Gln Ser His Cys 1 5
238PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(8) 23Cys Gly Asn Pro Ser Tyr Arg Cys 1 5
2420PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(20) 24Tyr Pro Cys Gly Gly Glu Ala Val Ala Gly
Val Ser Ser Val Arg Thr 1 5 10 15 Met Cys Ser Glu 20
2520PRTArtificial Sequencechemically synthesized; brain homing
peptidepeptide(1)..(20) 25Leu Asn Cys Asp Tyr Gln Gly Thr Asn Pro
Ala Thr Ser Val Ser Val 1 5 10 15 Pro Cys Thr Val 20
267PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(7) 26Cys Leu Pro Val Ala Ser Cys 1 5
277PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(7) 27Cys Gly Ala Arg Glu Met Cys 1 5
288PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(8) 28Cys Lys Gly Arg Ser Ser Ala Cys 1 5
298PRTArtificial Sequencechemically synthesized; kidney homing
peptide 29Cys Trp Ala Arg Ala Gln Gly Cys 1 5 308PRTArtificial
Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(8) 30Cys Leu Gly Arg Ser Ser Val Cys 1 5
318PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(8) 31Cys Thr Ser Pro Gly Gly Ser Cys 1 5
328PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(8) 32Cys Met Gly Arg Trp Arg Leu Cys 1 5
338PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(8) 33Cys Val Gly Glu Cys Gly Gly Cys 1 5
347PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(7) 34Cys Val Ala Trp Leu Asn Cys 1 5
357PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(7) 35Cys Arg Arg Phe Gln Asp Cys 1 5
367PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(7) 36Cys Leu Met Gly Val His Cys 1 5
378PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(8) 37Cys Lys Leu Leu Ser Gly Val Cys 1 5
388PRTArtificial Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(8) 38Cys Phe Val Gly His Asp Leu Cys 1 5
397PRTArtificial Sequencechemically synthesized; kidney homing
peptide 39Cys Arg Cys Leu Asn Val Cys 1 5 407PRTArtificial
Sequencechemically synthesized; kidney homing
peptidepeptide(1)..(7) 40Cys Lys Leu Met Gly Glu Cys 1 5
419PRTArtificial Sequencechemically synthesized; skin homing
peptidepeptide(1)..(9) 41Cys Ala Arg Ser Lys Asn Lys Asp Cys 1 5
426PRTArtificial Sequencechemically synthesized; skin homing
peptidepeptide(1)..(6) 42Cys Arg Lys Asp Lys Cys 1 5
4313PRTArtificial Sequencechemically synthesized; skin homing
peptidepeptide(1)..(13) 43Cys Val Ala Leu Cys Arg Glu Ala Cys Gly
Glu Gly Cys 1 5 10 4413PRTArtificial Sequencechemically
synthesized; skin homing peptidepeptide(1)..(13) 44Cys Ser Ser Gly
Cys Ser Lys Asn Cys Leu Glu Met Cys 1 5 10 458PRTArtificial
Sequencechemically synthesized; skin homing peptidepeptide(1)..(8)
45Cys Ile Gly Glu Val Glu Val Cys 1 5 469PRTArtificial
Sequencechemically synthesized; skin homing peptidepeptide(1)..(9)
46Cys Lys Trp Ser Arg Leu His Ser Cys 1 5 479PRTArtificial
Sequencechemically synthesized; skin homing peptidepeptide(1)..(9)
47Cys Trp Arg Gly Asp Arg Lys Ile Cys 1 5 489PRTArtificial
Sequencechemically synthesized; skin homing peptidepeptide(1)..(9)
48Cys Glu Arg Val Val Gly Ser Ser Cys 1 5 499PRTArtificial
Sequencechemically synthesized; skin homing peptidepeptide(1)..(9)
49Cys Leu Ala Lys Glu Asn Val Val Cys 1 5 5013PRTArtificial
Sequencechemically synthesized; lung homing peptidepeptide(1)..(13)
50Cys Gly Phe Glu Cys Val Arg Gln Cys Pro Glu Arg Cys 1 5 10
518PRTArtificial Sequencechemically synthesized; lung homing
peptidepeptide(1)..(8) 51Cys Gly Phe Glu Leu Glu Thr Cys 1 5
528PRTArtificial Sequencechemically synthesized; lung homing
peptidepeptide(1)..(8) 52Cys Thr Leu Arg Asp Arg Asn Cys 1 5
538PRTArtificial Sequencechemically synthesized; lung homing
peptidepeptide(1)..(8) 53Cys Ile Gly Glu Val Glu Val Cys 1 5
548PRTArtificial Sequencechemically synthesized; lung homing
peptidepeptide(1)..(8) 54Cys Thr Leu Arg Asp Arg Asn Cys 1 5
558PRTArtificial Sequencechemically synthesized; lung homing
peptide 55Cys Gly Lys Arg Tyr Arg Asn Cys 1 5 568PRTArtificial
Sequencechemically synthesized; lung homing peptide 56Cys Leu Arg
Pro Tyr Leu Asn Cys 1 5 5712PRTArtificial Sequencechemically
synthesized; lung homing peptidepeptide(1)..(12) 57Cys Thr Val Asn
Glu Ala Tyr Lys Thr Arg Met Cys 1 5 10 5812PRTArtificial
Sequencechemically synthesized; lung homing peptidepeptide(1)..(12)
58Cys Arg Leu Arg Ser Tyr Gly Thr Leu Ser Leu Cys 1 5 10
5912PRTArtificial Sequencechemically synthesized; lung homing
peptide 59Cys Arg Pro Trp His Asn Gln Ala His Thr Glu Cys 1 5 10
609PRTArtificial Sequencechemically synthesized; pancreas homing
peptidepeptide(1)..(9) 60Ser Trp Cys Glu Pro Gly Trp Cys Arg 1 5
617PRTArtificial Sequencechemically synthesized; pancreas homing
peptidepeptide(1)..(7) 61Cys Lys Ala Ala Lys Asn Lys 1 5
627PRTArtificial Sequencechemically synthesized; pancreas homing
peptidepeptide(1)..(7) 62Cys Lys Gly Ala Lys Ala Arg 1 5
638PRTArtificial Sequencechemically synthesized; pancreas homing
peptidepeptide(1)..(8) 63Val Gly Val Gly Glu Trp Ser Val 1 5
647PRTArtificial Sequencechemically synthesized; intestine homing
peptidepeptide(1)..(7) 64Tyr Ser Gly Lys Trp Gly Trp 1 5
657PRTArtificial Sequencechemically synthesized; uterus homing
peptidepeptide(1)..(7) 65Gly Leu Ser Gly Gly Arg Ser 1 5
667PRTArtificial Sequencechemically synthesized; adrenal gland
homing peptidepeptide(1)..(7) 66Leu Met Leu Pro Arg Ala Asp 1 5
677PRTArtificial Sequencechemically synthesized; adrenal gland
homing peptidepeptide(1)..(7) 67Leu Pro Arg Tyr Leu Leu Ser 1 5
689PRTArtificial Sequencechemically synthesized; retina homing
peptidepeptide(1)..(9) 68Cys Ser Cys Phe Arg Asp Val Cys Cys 1 5
699PRTArtificial Sequencechemically synthesized; retina homing
peptidepeptide(1)..(9) 69Cys Arg Asp Val Val Ser Val Ile Cys 1 5
707PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 70Tyr Ser Gly Lys Trp Gly Lys 1 5
718PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(8) 71Gly Ile Ser Ala Leu Val Leu Ser 1 5
727PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 72Ser Arg Arg Gln Pro Leu Ser 1 5
737PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 73Met Ser Pro Gln Leu Ala Thr 1 5
747PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 74Met Arg Arg Asp Glu Gln Arg 1 5
757PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 75Gln Val Arg Arg Val Pro Glu 1 5
767PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 76Val Arg Arg Gly Ser Pro Gln 1 5
777PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 77Gly Gly Arg Gly Ser Trp Glu 1 5
787PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 78Phe Arg Val Arg Gly Ser Pro 1 5
797PRTArtificial Sequencechemically synthesized; gut homing
peptidepeptide(1)..(7) 79Arg Val Arg Gly Pro Glu Arg 1 5
807PRTArtificial Sequencechemically synthesized; liver homing
peptidepeptide(1)..(7) 80Val Lys Ser Val Cys Arg Thr 1 5
817PRTArtificial Sequencechemically synthesized; liver homing
peptidepeptide(1)..(7) 81Trp Arg Gln Asn Met Pro Leu 1 5
827PRTArtificial Sequencechemically synthesized; liver homing
peptidepeptide(1)..(7) 82Ser Arg Arg Phe Val Gly Gly 1 5
837PRTArtificial Sequencechemically synthesized; liver homing
peptide 83Ala Leu Glu Arg Arg Ser Leu 1 5 847PRTArtificial
Sequencechemically synthesized; liver homing peptidepeptide(1)..(7)
84Ala Arg Arg Gly Trp Thr Leu 1 5 857PRTArtificial
Sequencechemically synthesized; prostate homing
peptidepeptide(1)..(7) 85Ser Met Ser Ile Ala Arg Leu 1 5
867PRTArtificial Sequencechemically synthesized; prostate homing
peptidepeptide(1)..(7) 86Val Ser Phe Leu Glu Tyr Arg 1 5
877PRTArtificial Sequencechemically synthesized; prostate homing
peptidepeptide(1)..(7) 87Arg Gly Arg Trp Leu Ala Leu 1 5
887PRTArtificial Sequencechemically synthesized; ovary homing
peptidepeptide(1)..(7) 88Glu Val Arg Ser Arg Leu Ser 1 5
897PRTArtificial Sequencechemically synthesized; ovary homing
peptidepeptide(1)..(7) 89Val Arg Ala Arg Leu Met Ser 1 5
907PRTArtificial Sequencechemically synthesized; ovary homing
peptidepeptide(1)..(7) 90Arg Val Gly Leu Val Ala Arg 1 5
917PRTArtificial Sequencechemically synthesized; ovary homing
peptidepeptide(1)..(7) 91Arg Val Arg Leu Val Asn Leu 1 5
925PRTArtificial Sequencechemically synthesized; clot
binding/homing peptidepeptide(1)..(5) 92Cys Arg Glu Lys Ala 1 5
935PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(5) 93Cys Arg Pro Pro Arg 1 5 949PRTArtificial
Sequencechemically synthesized; heart homing peptidepeptide(1)..(9)
94Cys Gly Arg Lys Ser Lys Thr Val Cys 1 5 956PRTArtificial
Sequencechemically synthesized; heart homing peptidepeptide(1)..(6)
95Cys Ala Arg Pro Ala Arg 1 5 966PRTArtificial Sequencechemically
synthesized; heart homing peptidepeptide(1)..(6) 96Cys Pro Lys Arg
Pro Arg 1 5 976PRTArtificial Sequencechemically synthesized; heart
homing peptidepeptide(1)..(6) 97Cys Lys Arg Ala Val Arg 1 5
989PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(9) 98Cys Arg Asn Ser Trp Lys Pro Asn Cys 1 5
995PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(5) 99Arg Gly Ser Ser Ser 1 5 1009PRTArtificial
Sequencechemically synthesized; heart homing peptidepeptide(1)..(9)
100Cys Arg Ser Thr Arg Ala Asn Pro Cys 1 5 1019PRTArtificial
Sequencechemically synthesized; heart homing peptidepeptide(1)..(9)
101Cys Pro Lys Thr Arg Arg Val Pro Cys 1 5 1029PRTArtificial
Sequencechemically synthesized; heart homing peptide 102Cys Ser Gly
Met Ala Arg Thr Lys Cys 1 5 1037PRTArtificial Sequencechemically
synthesized; heart homing peptidepeptide(1)..(7) 103Gly Gly Gly Val
Phe Trp Gln 1 5 1047PRTArtificial Sequencechemically synthesized;
heart homing peptide 104His Gly Arg Val Arg Pro His 1 5
1057PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(7) 105Val Val Leu Val Thr Ser Ser 1 5
1068PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(8) 106Cys Leu His Arg Gly Asn Ser Cys 1 5
10712PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(12) 107Cys Arg Ser Trp Asn Lys Ala Asp Asn Arg
Ser Cys 1 5 10 1089PRTArtificial Sequencechemically synthesized;
heart homing peptidepeptide(1)..(9) 108Cys Gly Arg Lys Ser Lys Thr
Val Cys 1 5 1096PRTArtificial Sequencechemically synthesized; heart
homing peptidepeptide(1)..(6) 109Cys Lys Arg Ala Val Arg 1 5
1109PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(9) 110Cys Arg Asn Ser Trp Lys Pro Asn Cys 1 5
1119PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(9) 111Cys Pro Lys Thr Arg Arg Val Pro Cys 1 5
1129PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(9) 112Cys Ser Gly Met Ala Arg Thr Lys Cys 1 5
1136PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(6) 113Cys Ala Arg Pro Ala Arg 1 5
1146PRTArtificial Sequencechemically synthesized; heart homing
peptidepeptide(1)..(6) 114Cys Pro Lys Arg Pro Arg 1 5
1155PRTArtificial Sequencechemically synthesized; tumor blood
vessel homing peptidepeptide(1)..(5) 115Cys Asn Gly Arg Cys 1 5
1169PRTArtificial Sequencechemically synthesized; homing
peptidepeptide(1)..(9) 116Cys Ser Arg Pro Arg Arg Ser Glu Cys 1 5
1179PRTArtificial Sequencechemically synthesized; homing
peptidepeptide(1)..(9) 117Cys Ser Arg Pro Arg Arg Ser Val Cys 1 5
1189PRTArtificial Sequencechemically synthesized; homing
peptidepeptide(1)..(9) 118Cys Ser Arg Pro Arg Arg Ser Trp Cys 1 5
11931PRTArtificial Sequencechemically synthesized; homing
peptidepeptide(1)..(31) 119Lys Asp Glu Pro Gln Arg Arg Ser Ala Arg
Leu Ser Ala Lys Pro Ala 1 5 10 15 Pro Pro Lys Pro Glu Pro Lys Pro
Lys Lys Ala Pro Ala Lys Lys 20 25 30 12010PRTArtificial
Sequencechemically synthesized; homing peptidepeptide(1)..(10)
120Pro Gln Arg Arg Ser Ala Arg Leu Ser Ala 1 5 10
12110PRTArtificial Sequencechemically synthesized; homing peptide
121Pro Lys Arg Arg Ser Ala Arg Leu Ser Ala 1 5 10 1227PRTArtificial
Sequencechemically synthesized; homing peptidepeptide(1)..(7)
122Cys Arg Gly Arg Arg Ser Thr 1 5 1239PRTArtificial
Sequencechemically synthesized; tumor homing peptidepeptide(1)..(9)
123Cys Ala Arg Ser Lys Asn Lys Asp Cys 1 5 1246PRTArtificial
Sequencechemically synthesized; tumor homing peptidepeptide(1)..(6)
124Cys Arg Lys Asp Lys Cys 1 5 12510PRTArtificial
Sequencechemically synthesized; tumor homing
peptidepeptide(1)..(10) 125Cys Gly Leu Ile Ile Gln Lys Asn Glu Cys
1 5 10 12610PRTArtificial Sequencechemically synthesized; tumor
homing peptidepeptide(1)..(10) 126Cys Asn Ala Gly Glu Ser Ser Lys
Asn Cys 1 5 10 1279PRTArtificial Sequencechemically synthesized;
tumor homing peptidepeptide(1)..(9) 127Cys Gly Asn Lys Arg Thr Arg
Gly Cys 1 5 1289PRTArtificial Sequencechemically synthesized; tumor
homing peptidepeptide(1)..(9) 128Cys Ala Arg Ser Lys Asn Lys Asp
Cys 1 5 1296PRTArtificial Sequencechemically synthesized; homing
peptidepeptide(1)..(6) 129Cys Arg Lys Asp Lys Cys 1 5
1304PRTArtificial Sequencechemically synthesized; internalization
sequenceMISC_FEATURE(1)..(1)X at position 1 can be either arginine,
lysine or histidineMISC_FEATURE(2)..(2)X at position 2 can be any
amino acidMISC_FEATURE(3)..(3)X at position 3 can be any amino
acidMISC_FEATURE(4)..(4)X at position 4 can be either arginine,
lysine or histidine 130Xaa Xaa Xaa Xaa 1 1315PRTArtificial
Sequencechemically synthesized; internalization
sequenceMISC_FEATURE(1)..(1)X at position 1 can be either arginine,
lysine or histidineMISC_FEATURE(2)..(2)X at position 2 can be any
amino acidMISC_FEATURE(3)..(3)X at position 3 can be any amino acid
131Xaa Xaa Xaa Lys Gly 1 5 1329PRTArtificial Sequencechemically
synthesized; tumor homing peptidepeptide(1)..(9) 132Cys Asn Arg Arg
Thr Lys Ala Gly Cys 1 5 1336PRTArtificial Sequencechemically
synthesized; tumor homing peptidepeptide(1)..(6) 133Cys Arg Ser Arg
Lys Gly 1 5 1349PRTArtificial Sequencechemically synthesized; tumor
homing peptidepeptide(1)..(9) 134Cys Arg Gly Asp Lys Gly Pro Asp
Cys 1 5 1355PRTArtificial Sequencechemically synthesized; control
peptidepeptide(1)..(5) 135Lys Ala Lys Glu Cys 1 5 1365PRTArtificial
Sequencechemically synthesized; homing peptidepeptide(1)..(5)
136Cys Arg Gly Asp Cys 1 5 13734PRTArtificial Sequencechemically
synthesized; tumor homing peptide 137Ala Lys Val Lys Asp Glu Pro
Gln Arg Arg Ser Ala Arg Leu Ser Ala 1 5 10 15 Lys Pro Ala Pro Pro
Lys Pro Glu Pro Lys Pro Lys Lys Ala Pro Ala 20 25 30 Lys Lys
* * * * *